LFA-1 antagonist compounds

ABSTRACT

The invention relates to novel compounds having formula (I)wherein Cy, X, Y, L and R1-6 are as defined herein. The compounds bind CD11/CD18 adhesion receptors such as Lymphocyte Function-associated Antigen-1 (LFA-1) and are therefore useful for treating disorders mediated by LFA-1 such as inflammation and autoimmune diseases.

This application claims priority to provisional application No.60/253,682 filed Nov. 28, 2000, the entire disclosure of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to novel compounds which bind CD11/CD18 adhesionreceptors, in particular Lymphocyte Function-associated Antigen-1(LFA-1) as well as pharmaceutical compositions containing thesecompounds which are useful for treating disorders mediated thereby.

BACKGROUND OF THE INVENTION

Inflammation

Human peripheral blood is composed principally of red blood cells,platelets and white blood cells or leukocytes. The family of leukocytesare further classified as neutrophils, lymphocytes (mostly B- and T-cellsubtypes), monocytes, eosinophils and basophils. Neutrophils,eosinophils and basophils are sometimes referred to as “granulocytes” or“polymorphonuclear (PMN) granulocytes” because of the appearance ofgranules in their cytoplasm and their multiple nuclei. Granulocytes andmonocytes are often classified as “phagocytes” because of their abilityto phagocytose or ingest micro-organisms and foreign mater referred togenerally as “antigens”. Monocytes are so called because of their largesingle nucleus and these cells may in turn become macrophages.Phagocytes are important in defending the host against a variety ofinfections and together with lymphocytes are also involved ininflammatory disorders. The neutrophil is the most common leukocytefound in human peripheral blood followed closely by the lymphocyte. In amicroliter of normal human peripheral blood, there are about 6,000leukocytes, of which about 4,000 are neutrophils, 1500 are lymphocytes,250 are monocytes, 150 are eosinophils and 25 are basophils.

During an inflammatory response peripheral blood leukocytes arerecruited to the site of inflammation or injury by a series of specificcellular interactions (see FIG. 1). The initiation and maintenance ofimmune functions are regulated by intercellular adhesive interactions aswell as signal transduction resulting from interactions betweenleukocytes and other cells. Leukocyte adhesion to vascular endotheliumand migration from the circulation to sites of inflammation is acritical step in the inflammatory response (FIG. 1). T-cell lymphocyteimmune recognition requires the interaction of the T-cell receptor withantigen (in combination with the major histocompatibility complex) aswell as adhesion receptors, which promote attachment of T-cells toantigen-presenting cells and transduce signals for T-cell activation.The lymphocyte function associated antigen-1 (LFA-1) has been identifiedas the major integrin that mediates lymphocyte adhesion and activationleading to a normal immune response, as well as several pathologicalstates (Springer, T. A., Nature 346:425-434 (1990)). Intercellularadhesion molecules (ICAM)-1, -2, and -3, members of the immunoglobulinsuperfamily, are ligands for LFA-1 found on endothelium, leukocytes andother cell types. The binding of LFA-1 to ICAMs mediate a range oflymphocyte functions including lymphokine production of helper T-cellsin response to antigen presenting cells, T-lymphocyte mediated targetcells lysis, natural killing of tumor cells, and immunoglobulinproduction through T-cell-B-cell interactions. Thus, many facets oflymphocyte function involve the interaction of the LFA-1 integrin andits ICAM ligands. These LFA-1:ICAM mediated interactions have beendirectly implicated in numerous inflammatory disease states including;graft rejection, dermatitis, psoriasis, asthma and rheumatoid arthritis.

While LFA-1 (CD11a/CD18) on lymphocytes plays a key role in chronicinflammation and immune responses, other members of the leukocyteintegrin family (CD11b/CD18, CD11c/CD18 and CD11d/CD18) also playimportant roles on other leukocytes, such as granulocytes and monocytes,particularly in early response to infective agents and in acuteinflammatory response.

The primary function of polymorphonuclear leukocytes, derived from theneutrophil, eosinophil and basophil lineage, is to sense inflammatorystimuli and to emigrate across the endothelial barrier and carry outscavenger function as a first line of host defense. The integrinMac-1(CD11b/CD18) is rapidly upregulated on these cells upon activationand binding to its multiple ligands which results in the release ofoxygen derived free radicals, protease's and phospholipases. In certainchronic inflammatory states this recruitment is improperly regulatedresulting in significant cellular and tissue injury. (Harlan, J. M.,Acta Med Scand Suppl., 715:123 (1987); Weiss, S., New England J. ofMed., 320:365 (1989)). LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) The(CD11/CD18) family of adhesion receptor molecules comprises four highlyrelated cell surface glycoproteins; LFA-1 (CD11a/CD18), Mac-1(CD11b/CD18), p150.95 (CD11c/CD18) and (CD11d/CD18). LFA-1 is present onthe surface of all mature leukocytes except a subset of macrophages andis considered the major lymphoid integrin. The expression of Mac-1,p150.95 and CD11d/CD18 is predominantly confined to cells of the myeloidlineage (which include neutrophils, monocytes, macrophage and mastcells). Functional studies have suggested that LFA-1 interacts withseveral ligands, including ICAM-1 (Rothleinet al., J. Immunol.137:1270-1274 (1986), ICAM-2, (Staunton et al., Nature 339:361-364(1989)), ICAM-3 (Fawcett et al., Nature 360:481-484 (1992); Vezeux etal., Nature 360:485-488, (1992); de Fougerolles and Springer, J. Exp.Med. 175:185-190 (1990)) and Telencephalin (Tian et al., J. Immunol.158:928-936 (1997)).

The CD11/CD18 family is related structurally and genetically to thelarger integrin family of receptors that modulate cell adhesiveinteractions, which include; embryogenesis, adhesion to extracellularsubstrates, and cell differentiation (Hynes, R. O., Cell 48:549-554(1987); Kishimotoet al., Adv. Immunol. 46:149-182 (1989); Kishimotoetal., Cell 48:681-690 (1987); Ruoslahtiet al., Science 238:491-497(1987).

Integrins are a class of membrane-spanning heterodimers comprising an αsubunit in noncovalent association with a β subunit. The β subunits aregenerally capable of association with more than one α subunit and theheterodimers sharing a common β subunit have been classified assubfamilies within the integrin population (Larson and Springer,“Structure and function of leukocyte integrins,” Immunol. Rev114:181-217 (1990)).

The integrin molecules of the CD11/CD18 family, and their cellularligands, have been found to mediate a variety of cell—cell interactions,especially in inflammation. These proteins have been demonstrated to becritical for adhesive functions in the immune system (Kishimotoet al.,Adv. Immunol. 46:149-182 (1989)). Monoclonal antibodies to LFA-1 havebeen shown to block leukocyte adhesion to endothelial cells (Dustin etal., J. Cell. Biol. 107:321-331 (1988); Smith et al., J. Clin. Invest.83:2008-2017 (1989)) and to inhibit T-cell activation (Kuypers et al.,Res. Immnunol., 140:461 (1989)), conjugate formation required forantigen-specific CTL killing (Kishimotoet al., Adv. Immunol. 46:149-182(1989)), T. cell proliferation (Davignonet al., J. Immunol. 127:590-595(1981)) and NK cell killing (Krenskyet al., J. Immunol. 131:611-616(1983)). ICAMs

ICAM-1 (CD54) is a cell surface adhesion receptor that is a member ofthe immunoglobulin protein super-family (Rothleinet al., J. Immunol.137:1270-1274 (1986); Stauntonet al., Cell 52:925-933 (1988). Members ofthis superfamily are characterized by the presence of one or more Ighomology regions, each consisting of a disulfide-bridged loop that has anumber of anti-parallel β-pleated strands arranged in two sheets. Threetypes of homology regions have been identified, each with a typicallength and having a consensus sequence of amino acid residues locatedbetween the cysteines of the disulfide bond (Williams, A. F. et al. AnnRev. Immunol. 6:381-405 (1988); Hunkapillar, T. et al. Adv. Immunol.44:1-63 (1989). ICAM-1 is expressed on a variety of hematopoietic andnon-hematopoietic cells and is upregulated at sites of inflammation by avariety of inflammatory mediators (Dustin et al., J. Immunol.,137:256-254 (1986)). ICAM-1 is a 90,000-110,000 M_(r) glycoprotein witha low messenger RNA levels and moderate surface expression onunstimulated endothelial cells. LPS, IL-1 and TNF strongly upregulateICAM-1 mRNA and surface expression with peak expression at approximately18-24 hours (Dustinet al., J. Cell. Biol. 107:321-331 (1988); Stauntonetal., Cell 52:925-933 (1988)). ICAM-1 has five extracellular Ig likedomains (designated Domains 1, 2, 3, 4 and 5 or D1, D2, D3, D4 and D5)and an intracellular or cytoplasmic domain. The structures and sequenceof the domains is described by Staunton et al. (Cell 52:925-933 (1988)).

ICAM-1 was defined originally as a counter-receptor for LFA-1 (Springeret al., Ann. Rev. Immunol, 5:223-252 (1987); MarlinCell 51:813-819(1987); Simmonset al., Nature 331:624-627 (1988); Staunton Nature339:61-64 (1989); Stauntonet al., Cell 52:925-933 (1988)). TheLFA-1/ICAM-1 interaction is known to be at least partially responsiblefor lymphocyte adhesion (Dustinet al., J. Cell. Biol. 107:321-331(1988); Mentzeret al., J. Cell. Physiol. 126:285-290 (1986)), monocyteadhesion (Amaout et al., J. Cell Physiol. 137:305 (1988); Mentzeret al.,J. Cell. Physiol. 130:410-415 (1987); te Veldeet al., Immunology61:261-267 (1987)), and neutrophil adhesion (Loet al., J. Immunol.143(10):3325-3329 (1989); Smith et al., J. Clin. Invest. 83:2008-2017(1989)) to endothelial cells. Through the development of functionblocking monoclonal antibodies to ICAM-1 additional ligands for LFA-1were identified, ICAM-2 and ICAM-3 (Simmons, Cancer Surveys 24, CellAdhesion and Cancer, 1995) that mediate the adhesion of lymphocytes toother leukocytes as well as non-hematopoietic cells. Interactions ofLFA-1 with ICAM-2 are thought to mediate natural killer cell activity(Helander et al., Nature 382:265-267 (1996)) and ICAM-3 binding isthought to play a role in lymphocyte activation and the initiation ofthe immune response (Simmons, ibid). The precise role of these ligandsin normal and aberrant immune responses remains to be defined.

Disorders Mediated by T Lymphocytes

Function blocking monoclonal antibodies have shown that LFA-1 isimportant in T-lymphocyte-mediated killing, T-helper lymphocyteresponses, natural killing, and antibody-dependent killing (Springer etal., Ann. Rev. Immunol 5:223-252 (1987)). Adhesion to the target cell aswell as activation and signaling are steps that are blocked byantibodies against LFA-1.

Many disorders and diseases are mediated through T lymphocytes andtreatment of these diseases have been addressed through many routes.Rheumatoid arthritis (RA) is one such disorder. Current therapy for RAincludes bed rest, application of heat, and drugs. Salicylate is thecurrently preferred treatment drug, particularly as other alternativessuch as immunosuppressive agents and adrenocorticosteroids can causegreater morbidity than the underlying disease itself. Nonsteroidalanti-inflammatory drugs are available, and many of them have effectiveanalgesic, anti-pyretic and anti-inflammatory activity in RA patients.These include cyclosporin, indomethacin, phenylbutazone, phenylaceticacid derivatives such as ibuprofen and fenoprofen, naphthalene aceticacids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids(sulindac), halogenated anthranilic acid (meclofenamate sodium),piroxicam, and diflunisal. Other drugs for use in RA includeanti-malarials such as chloroquine, gold salts and penicillamine. Thesealternatives frequently produce severe side effects, including retinallesions and kidney and bone marrow toxicity. Immunosuppressive agentssuch as methotrexate have been used only in the treatment of severe andunremitting RA because of their toxicity. Corticosteroids also areresponsible for undesirable side effects (e.g., cataracts, osteoporosis,and Cushing's disease syndrome) and are not well tolerated in many RApatients.

Another disorder mediated by T lymphocytes is host rejection of graftsafter transplantation. Attempts to prolong the survival of transplantedallografts and xenografts, or to prevent host versus graft rejection,both in experimental models and in medical practice, have centeredmainly on the suppression of the immune apparatus of the host/recipient.This treatment has as its aim preventive immunosuppression and/ortreatment of graft rejection. Examples of agents used for preventiveimmunosuppression include cytotoxic drugs, anti-metabolites,corticosteroids, and anti-lymphocytic serum. Nonspecificimmunosuppressive agents found particularly effective in preventiveimmunosuppression (azathioprine, bromocryptine, methylprednisolone,prednisone, and most recently, cyclosporin A) have significantlyimproved the clinical success of transplantation. The nephrotoxicity ofcyclosporin A after renal transplantation has been reduced byco-administration of steroids such as prednisolone, or prednisolone inconjunction with azathioprine. In addition, kidneys have been graftedsuccessfully using anti-lymphocyte globulin followed by cyclosporin A.Another protocol being evaluated is total lymphoid irradiation of therecipient prior to transplantation followed by minimal immunosuppressionafter transplantation.

Treatment of rejection has involved use of steroids,2-amino-6-aryl-5-substituted pyrimidines, heterologous anti-lymphocyteglobulin, and monoclonal antibodies to various leukocyte populations,including OKT-3. See generally J. Pediatrics, 111: 1004-1007 (1987), andspecifically U.S. Pat. No. 4,665,077.

The principal complication of immunosuppressive drugs is infections.Additionally, systemic immunosuppression is accompanied by undesirabletoxic effects (e.g., nephrotoxicity when cyclosporin A is used afterrenal transplantation) and reduction in the level of the hemopoieticstem cells. Immunosuppressive drugs may also lead to obesity, poor woundhealing, steroid hyperglycemia, steroid psychosis, leukopenia,gastrointestinal bleeding, lymphoma, and hypertension.

In view of these complications, transplantation immunologists havesought methods for suppressing immune responsiveness in anantigen-specific manner (so that only the response to the donoralloantigen would be lost). In addition, physicians specializing inautoimmune disease strive for methods to suppress autoimmuneresponsiveness so that only the response to the self-antigen is lost.Such specific immunosuppression generally has been achieved by modifyingeither the antigenicity of the tissue to be grafted or the specificcells capable of mediating rejection. In certain instances, whetherimmunity or tolerance will be induced depends on the manner in which theantigen is presented to the immune system.

Pretreating the allograft tissues by growth in tissue culture beforetransplantation has been found in two murine model systems to lead topermanent acceptance across MHC barriers. Lafferty et al.,Transplantation, 22:138-149 (1976); Bowen et al., Lancet, 2:585-586(1979). It has been hypothesized that such treatment results in thedepletion of passenger lymphoid cells and thus the absence of astimulator cell population necessary for tissue immunogenicity. Laffertyet al., Annu. Rev. Immunol., 1:143 (1983). See also Lafferty et al.,Science, 188:259-261 (1975) (thyroid held in organ culture), and Goreset al., J. Immunol., 137:1482-1485 (1986) and Faustman et al., Proc.Natl. Acad. Sci. U.S.A., 78: 5156-5159 (1981) (islet cells treated withmurine anti-Ia antisera and complement before transplantation). Also,thyroids taken from donor animals pretreated with lymphocytotoxic drugsand gamma radiation and cultured for ten days in vitro were not rejectedby any normal allogeneic recipient (Gose and Bach, J. Exp. Med.,149:1254-1259 (1979)). All of these techniques involve depletion orremoval of donor lymphocyte cells.

In some models such as vascular and kidney grafts, there exists acorrelation between Class II matching and prolonged allograft survival,a correlation not present in skin grafts (Pescovitz et al., J. Exp.Med., 160:1495-1508 (1984); Conti et al., Transplant. Proc., 19: 652-654(1987)). Therefore, donor-recipient HLA matching has been utilized.Additionally, blood transfusions prior to transplantation have beenfound to be effective (Opelz et al., Transplant. Proc., 4: 253 (1973);Persijn et al., Transplant. Proc., 23:396 (1979)). The combination ofblood transfusion before transplantation, donor-recipient HLA matching,and immunosuppression therapy (cyclosporin A) after transplantation wasfound to improve significantly the rate of graft survival, and theeffects were found to be additive (Opelz et al., Transplant. Proc.,17:2179 (1985)).

The transplantation response may also be modified by antibodies directedat immune receptors for MHC antigens (Bluestone et al., Immunol. Rev90:5-27 (1986)). Further, graft survival can be prolonged in thepresence of antigraft antibodies, which lead to a host reaction that inturn produces specific immunosuppression (Lancaster et al., Nature, 315:336-337 (1985)). The immune response of the host to MHC antigens may bemodified specifically by using bone marrow transplantation as apreparative procedure for organ grafting. Thus, anti-T-cell monoclonalantibodies are used to deplete mature T-cells from the donor marrowinoculum to allow bone marrow transplantation without incurringgraft-versus-host disease (Mueller-Ruchholtz et al., Transplant Proc.,8:537-541 (1976)). In addition, elements of the host's lymphoid cellsthat remain for bone marrow transplantation solve the problem ofimmunoincompetence occurring when fully allogeneic transplants are used.

As shown in FIG. 1, lymphocyte adherence to endothelium is a key eventin the process of inflammation. There are at least three known pathwaysof lymphocyte adherence to endothelium, depending on the activationstate of the T-cell and the endothelial cell. T-cell immune recognitionrequires the contribution of the T-cell receptor as well as adhesionreceptors, which promote attachment of—cells to antigen-presenting cellsand transduce regulatory signals for T-cell activation. The lymphocytefunction associated (LFA) antigen-1 (LFA-1, CD11a/CD18, □ α_(Lβ) 2:where α_(L) is CD11a and β₂ is CD18) has been identified as the majorintegrin receptor on lymphocytes involved in these cell adherenceinteractions leading to several pathological states. ICAM-1, theendothelial cell immunoglobulin-like adhesion molecule, is a knownligand for LFA-1 and is implicated directly in graft rejection,psoriasis, and arthritis.

LFA-1 is required for a range of leukocyte functions, includinglymphokine production of helper T-cells in response toantigen-presenting cells, killer T-cell-mediated target cell lysis, andimmunoglobulin production through T-cell/B-cell interactions. Activationof antigen receptors on T-cells and B-cells allows LFA-1 to bind itsligand with higher affinity.

Monoclonal antibodies (MAbs) directed against LFA-1 led to the initialidentification and investigation of the function of LFA-1 (Davignon etal., J. Immunol., 127:590 (1981)). LFA-1 is present only on leukocytes(Krenskey et al., J. Immunol., 131:611 (1983)), and ICAM-1 isdistributed on activated leukocytes, dermal fibroblasts, and endothelium(Dustin et al., J. Immunol. 137:245 (1986)).

Previous studies have investigated the effects of anti-CD11a MAbs onmany T-cell-dependent immune functions in vitro and a limited number ofimmune responses in vivo. In vitro, anti-CD11a MAbs inhibit T-cellactivation (Kuypers et al., Res. Immunol., 140:461 (1989)),T-cell-dependent B-cell proliferation and differentiation (Davignon etal., supra; Fischer et al., J. Immunol., 136:3198 (1986)), target celllysis by cytotoxic T-lymphocytes (Krensky et al., supra), formation ofimmune conjugates (Sanders et al., J. Immunol., 137:2395 (1986); Mentzeret al., J. Immunol., 135:9 (1985)), and the adhesion of T-cells tovascular endothelium (Lo et al., J. Immunol., 143:3325 (1989)). Also,the antibody 5C6 directed against CD11b/CD18 was found to preventintra-islet infiltration by both macrophages and T cells and to inhibitdevelopment of insulin-dependent diabetes mellitis in mice (Hutchings etal., Nature, 348: 639 (1990))

The observation that LFA-1:ICAM-1 interaction is necessary to optimizeT-cell function in vitro, and that anti-CD11a MAbs induce tolerance toprotein antigens (Benjamin et al., Eur. J. Immunol., 18:1079 (1988)) andprolongs tumor graft survival in mice (Heagy et al., Transplantation,37: 520-523 (1984)) was the basis for testing the MAbs to thesemolecules for prevention of graft rejection in humans.

Experiments have also been carried out in primates. For example, basedon experiments in monkeys it has been suggested that a MAb directedagainst ICAM-1 can prevent or even reverse kidney graft rejection(Cosimi et al., “Immunosuppression of Cynomolgus Recipients of RenalAllografts by R6.5, a Monoclonal Antibody to Intercellular AdhesionMolecule-1,” in Springer et al. (eds.), Leukocyte Adhesion Molecules NewYork: Springer, (1988), p. 274; Cosimi et al., J. Immunology,144:4604-4612 (1990)). Furthermore, the in vivo administration ofanti-CD11a MAb to cynomolgus monkeys prolonged skin allograft survival(Berlin et al., Transplantation, 53: 840-849 (1992)).

The first successful use of a rat anti-murine CD11a antibody (25-3;IgG1) in children with inherited disease to prevent the rejection ofbone-marrow-mismatched haploidentical grafts was reported by Fischer etal., Lancet, 2: 1058 (1986). Minimal side effects were observed. Seealso Fischer et al., Blood, 77: 249 (1991); van Dijken et al.,Transplantation, 49:882 (1990); and Perez et al., Bone MarrowTransplantation, 4:379 (1989). Furthermore, the antibody 25-3 waseffective in controlling steroid-resistant acute graft-versus-hostdisease in humans (Stoppa et al., Transplant. Int., 4:3-7 (1991)).

However, these results were not reproducible in leukemic adult graftingwith this MAb (Maraninchi et al., Bone Marrow Transplant, 4:147-150(1989)), or with an anti-CD18 MAb, directed against the invariant chainof LFA-1, in another pilot study (Baume et al., Transplantation, 47: 472(1989)). Furthermore, a rat anti-murine CD11a MAb, 25-3, was unable tocontrol the course of acute rejection in human kidney transplantation(LeMauff et al., Transplantation, 52: 291 (1991)).

A review of the use of monoclonal antibodies in human transplantation isprovided by Dantal and Soulillou, Current Opinion in Immunology,3:740-747 (1991). An earlier report showed that brief treatment witheither anti-LFA-1 or anti-ICAM-1 MAbs minimally prolonged the survivalof primarily vascularized heterotopic heart allografts in mice (Isobe etal., Science, 255:1125 (1992)). However, combined treatment with bothMAbs was required to achieve long-term graft survival in this model.

Independently, it was shown that treatm7ent with anti-LFA-1 MAb alonepotently and effectively prolongs the survival of heterotopic(ear-pinnae) nonprimarily vascularized mouse heart grafts using amaximum dose of 4 mg/kg/day and treatment once a week after a daily dose(Nakakura et al., J. Heart Lung Transplant., 11:223 (1992)).Nonprimarily vascularized heart allografts are more immunogenic and moreresistant to prolongation of survival by MAbs than primarilyvascularized heart allografts (Warren et al., Transplant. Proc., 5:717(1973); Trager et al., Transplantation, 47:587 (1989)). The latterreference discusses treatment with L3T4 antibodies using a high initialdose and a lower subsequent dose.

Another study on treating a sclerosis-type disease in rodents usingsimilar antibodies to those used by Nakakura et al., supra, is reportedby Yednock et al., Nature, 356:63-66 (1992). Additional disclosures onthe use of anti-LFA-1 antibodies and ICAM-1, ICAM-2, and ICAM-3 andtheir antibodies to treat LFA-1-mediated disorders include WO 91/18011published Nov. 28, 1991, WO 91/16928 published Nov. 14, 1991, WO91/16927 published Nov. 14, 1991, Can. Pat. Appln. 2,008,368 publishedJun. 13, 1991, WO 90/03400, WO 90/15076 published Dec. 13, 1990, WO90/10652 published Sep. 20, 1990, EP 387,668 published Sep. 19, 1990, WO90/08187 published Jul. 26, 1990, WO 90/13281, WO 90/13316, WO 90/13281,WO 93/06864, WO 93/21953, WO 93/13210, WO 94/11400, EP 379,904 publishedAug. 1, 1990, EP 346,078 published Dec. 13, 1989, U.S. Pat. Nos.5,002,869, 5,071,964, 5,209,928, 5,223,396, 5,235,049, 5,284,931,5,288,854, 5,354,659, Australian Pat. Appln. 15518/88 published Nov. 10,1988, EP 289,949 published Nov. 9, 1988, and EP 303,692 published Feb.22, 1989, EP 365,837, EP 314,863, EP 319,815, EP 468, 257, EP 362,526,EP 362, 531, EP 438,310.

Other disclosures on the use of LFA-1 and ICAM peptide fragments andantagonists include; U.S. Pat. Nos. 5,149,780, 5,288,854, 5,340,800,5,424,399, 5,470,953, WO 90/03400, WO 90/13316, WO 90/10652, WO91/19511, WO 92/03473, WO 94/11400, WO 95/28170, JP 4193895, EP 314,863,EP 362,526 and EP 362,531.

The above methods successfully utilizing anti-LFA-1 or anti-ICAM-1antibodies, LFA-1 or ICAM-1 peptides, fragments or peptide antagonistsrepresent an improvement over traditional immunosuppressive drugtherapy. These studies demonstrate that LFA-1 and ICAM-1 are appropriatetargets for antagonism. There is a need in the art to better treatdisorders that are mediated by LFA-1 including autoimmune diseases,graft vs. host or host vs. graft rejection, and T-cell inflammatoryresponses, so as to minimize side effects and sustain specific toleranceto self- or xenoantigens.

There is also a need in the art to provide a non-peptide antagonists tothe LFA-1: ICAM-1 interaction.

Albumin is an abundant plasma protein which is responsible for thetransport of fatty acids. However, albumin also binds and perturbs thepharmacokinetics of a wide range of drug compounds. Accordingly, asignificant factor in the pharmacological profile of any drug is itsbinding characteristics with respect to serum plasma proteins such asalbumin. A drug compound may have such great affinity for plasmaproteins that it is not be available in serum to interact with itstarget tissue, cell or protein. For example, a compound for which 99%binds to plasma protein upon administration will have half theconcentration available in plasma to interact with its target than acompound which binds only 98%. Accordingly it would be desirable toprovide LFA antagonist compounds which have low serum plasma proteinbinding affinity.

SUMMARY OF THE INVENTION

In an aspect of the present invention, there is provided novel compoundsof formula (I)

wherein

Cy is a non-aromatic carbocycle or heterocycle optionally substitutedwith hydroxyl, mercapto, thioalkyl, halogen, oxo, thio, amino,aminoalkyl, amidine, guanidine, nitro, alkyl, alkoxy or acyl;

X is a divalent hydrocarbon chain optionally substituted with hydroxyl,mercapto, halogen, amino, aminoalkyl, nitro, oxo or thio and optionallyinterrupted with N, O, S, SO or SO₂;

Y is a carbocycle or heterocycle optionally substituted with hydroxyl,mercapto, halogen, oxo, thio, a hydrocarbon, a halo-substitutedhydrocarbon, amino, amidine, guanidine, cyano, nitro, alkoxy or acyl;

L is a bond or a divalent hydrocarbon optionally having one or morecarbon atoms replaced with N, O, S, SO or SO₂ and optionally beingsubstituted with hydroxyl, halogen oxo or thio; or three carbon atoms ofthe hydrocarbon are replaced with an amino acid residue;

R₁ is H, OH, amino, O-carbocycle or alkoxy optionally substituted withamino, a carbocycle or a heterocycle;

R₂₋₅ are independently H, hydroxyl, mercapto, halogen, cyano, amino,amidine, guanidine, nitro or alkoxy; or R₃ and R₄ together form a fusedcarbocycle or heterocycle optionally substituted with hydroxyl, halogen,oxo, thio, amino, amidine, guanidine or alkoxy;

R₆ is H or a hydrocarbon chain optionally substituted with a carbocycleor a heterocycle; and

salts, solvates and hydrates thereof;

with the proviso that when Y is phenyl, R₂, R₄ and R₅ are H, R₃ is Cland R₁ is OH then X is other than cyclohexyl.

In another aspect of the invention, there is provided pharmaceuticalcompositions comprising a compound of the invention and apharmaceutically acceptable carrier.

In another aspect of the invention, there is provided a method oftreating a disease or condition mediated by LFA-1 in a mammal comprisingadministering to said mammal an effective amount of a compound of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel compounds of formula (I)

wherein Cy, X, Y, L and R₁₋₆ are as defined herein. Compounds of theinvention exhibit reduced plasma protein binding affinity by virtue of anon-aromatic ring at substituent Cy in comparison to those having anaromatic ring at this portion of the molecule.

The term “non-aromatic” refers to carbocycle or heterocycle rings thatdo not have the properties which define aromaticity. For aromaticity, aring must be planar, have p-orbitals that are perpendicular to the planeof the ring at each ring atom and satisfy the Huckel rule where thenumber of pi electrons in the ring is (4n+2) wherein n is an integer(i.e. the number of pi electrons is 2, 6, 10 or 14). Non-aromatic ringsprovided herein do not satisfy one or all of these criteria foraromaticity.

The term “alkoxy” as used herein includes saturated, i.e. O-alkyl, andunsaturated, i.e. O-alkenyl and O-alkynyl, group. Exemplary alkoxygroups include methoxy, ethoxy, propoxy, butoxy, i-butoxy, s-butoxy,t-butoxy, pentyloxy and hexyloxy.

The term “amino” refers to a primary (—NH₂), secondary (—NHR), tertiary(—N(R)₂) or quaternary (—N⁺(R)₄) amine wherein R is a hydrocarbon chain,hydroxy, a carbocycle, a heterocycle or a hydrocarbon chain substitutedwith a carbocycle or heterocycle.

The term “amino acid” refers to naturally and non-naturally occurringα-(alpha), β-(beta), D- and L-amino acid residues. Non-natural aminoacids include those having side chains other than those occurring innature.

By “carboxyl” is meant herein to be a free acid —COOH as well as estersthereof such as alkyl, aryl and aralkyl esters. Preferred esters aremethyl, ethyl, propyl, butyl, i-butyl, s-butyl and t-butyl esters.

The term “carbocycle” refers to a mono-, bi- or tri-cyclic carbon ringor ring system having 4-16 members (including bridged) which issaturated, unsaturated or partially unsaturated including aromatic(aryl) ring systems (unless specified as non-aromatic). Preferrednon-aromatic carbocyclic rings include cyclopropyl, cyclopropenyl,cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl andcyclohexenyl. Preferred aromatic carbocyclic rings include phenyl andnaphthyl.

The term “heterocycle” refers to a mono-, bi- or tri-cyclic ring systemhaving 5-16 members wherein at least one ring atom is a heteroatom (i.e.N, O and S as well as SO, or SO₂). The ring system is saturated,unsaturated or partially unsaturated and may be aromatic (unlessspecified as non-aromatic). Exemplary heterocycles include piperidine,piperazine, pyridine, pyrazine, pyrimidine, pyridazine, morpholine,pyran, pyrole, furan, thiophene (thienyl), imidazole, pyrazole,thiazole, isothiazole, dithiazole, oxazole, isoxazole, dioxazole,thiadiazole, oxadiazole, tetrazole, triazole, thiatriazole, oxatriazole,thiadiazole, oxadiazole, purine and benzofused derivatives thereof.

The term “hydrocarbon chain” refers to saturated, unsaturated, linear orbranched carbon chains i.e. alkyl, alkenyl and alkynyl. Preferredhydrocarbon chains incorporate 1-12 carbon atoms, more preferably 1-6and most preferably 1-4 carbon atoms i.e. methyl, ethyl, propyl, butyland allyl.

The phrase “optionally substituted with” is understood to mean, unlessotherwise stated, that one or more of the specified substituents iscovalently attached to the substituted moiety. When more than one, thesubstituents may be the same or different group.

Cy is a non-aromatic carbocycle or heterocycle optionally substitutedwith hydroxyl (—OH), mercapto (—SH), thioalkyl, halogen (e.g. F, Cl, Br,I), oxo (═O), thio (═S), amino, aminoalkyl, amidine (—C(NH)—NH₂),guanidine (—NH₂—C(NH)—NH₂), nitro, alkyl or alkoxy. In a particularembodiment, Cy is a 3-5 member ring. In a preferred embodiment, Cy is a5- or 6-member non-aromatic heterocycle optionally substituted withhydroxyl, mercapto, halogen (preferably F or Cl), oxo (═O), thio (═S),amino, amidine, guanidine, nitro, alkyl or alkoxy. In a more preferredembodiment, Cy is a 5-member non-aromatic heterocycle optionallysubstituted with hydroxyl, oxo, thio, Cl, C₁₋₄ alkyl (preferablymethyl), or C₁₋₄ alkanoyl (preferably acetyl, propanoyl or butanoyl).More preferably the non-aromatic heterocycle comprises one orheteroatoms (N, O or S) and is optionally substituted with hydroxyl,oxo, mercapto, thio, methyl, acetyl, propanoyl or butyl. In particularembodiments the non-aromatic heterocycle comprises at least one nitrogenatom that is optionally substituted with methyl or acetyl. In aparticularly preferred embodiment, the non-aromatic heterocycle isselected from the group consisting of piperidine, piperazine,morpholine, tetrahydrofuran, tetrahydrothiophene, oxazolidine,thiazolidine optionally substituted with hydroxy, oxo, mercapto, thio,alkyl or alkanoyl. In a most preferred embodiment Cy is a non-aromaticheterocycle selected from the group consisting of tetrahydrofuran-2-yl,thiazolidin-5-yl, thiazolidin-2-one-5-yl, and thiazolidin-2-thione-5-yland cyclopropapyrrolidine.

In another preferred embodiment Cy is a 3-6 member carbocycle optionallysubstituted with hydroxyl, mercapto, halogen, oxo, thio, amino, amidine,guanidine, alkyl, alkoxy or acyl. In a particular embodiment thecarbocycle is saturated or partially unsaturated. In particularembodiments Cy is a carbocycle selected from the group consisting ofcyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclohexyl and cyclohexenyl.

X is a C₁₋₅ divalent hydrocarbon linker optionally having one or morecarbon atoms replaced with N, O, S, SO or SO₂ and optionally beingsubstituted with hydroxyl, mercapto, halogen, amino, aminoalkyl, nitro,oxo or thio. In a preferred embodiment X will have at least one carbonatom. Replacements and substitutions may form an amide moiety (—NRC(O)—or —C(O)NR—) within the hydrocarbon chain or at either or both ends.Other moieties include sulfonamide (—NRSO₂— or —SO₂NR), acyl, ether,thioether and amine. In a particularly preferred embodiment X is thegroup —CH₂—NR₆—C(O)— wherein the carbonyl —C(O)— portion thereof isadjacent (i.e. covalently bound) to Cy and R₆ is alkyl i.e. methyl andmore preferably H.

Y is a carbocycle or heterocycle optionally substituted with hydroxyl,mercapto, halogen, oxo, thio, a hydrocarbon, a halo-substitutedhydrocarbon, amino, amidine, guanidine, cyano, nitro, alkoxy or acyl. Inparticular embodiment, Y is aryl or heteroaryl optionally substitutedwith halogen or hydroxyl. In a particularly preferred embodiment, Y isphenyl, furan-2-yl, thiophene-2-yl, phenyl substituted with a halogen(preferably Cl) or hydroxyl, preferably at the meta position.

L is a divalent hydrocarbon optionally having one or more carbon atomsreplaced with N, O, S, SO or SO₂ and optionally being substituted withhydroxyl, halogen oxo, or thio; or three carbon atoms of the hydrocarbonare replaced with an amino acid residue. Preferably L is less than 10atoms in length and more preferably 5 or less and most preferably 5 or 3atoms in length. In particular embodiments, L is selected from the groupconsisting of —CH═CH—C(O)—NR₆—CH₂—, —CH₂—NR₆—C(O)—, —C(O)—NR₆—CH₂—,—CH(OH)—(CH₂)₂—, —(CH₂)₂—CH(OH)—, —(CH₂)₃—, —C(O)—NR₆—CH (R₇)—C(O)—NR₆—,—NR₆—C(O)—CH(R₇)—NR₆—C(O)—, —CH(OH)—CH₂—O— and —CH(OH)—CF₂—CH₂— whereineach R₆ is independently H or alkyl and R₇ is an amino acid side chain.Preferred amino acid side chains include non-naturally occurring sidechains such as phenyl or naturally occurring side chains. Preferred sidechains are those from Phe, Tyr, Ala, Gln and Asn. In a preferredembodiments L is —CH═CH—C(O)—NR₆—CH₂— wherein the —CH═CH— moiety thereofis adjacent (i.e. covalently bound) to Y. In another preferredembodiment, L is —CH₂— NR₆—C(O)— wherein the methylene moiety (—CH₂—)thereof is adjacent to Y.

R₁ is H, OH, amino, O-carbocycle or alkoxy optionally substituted withamino, a carbocycle or a heterocycle. In a preferred embodiment, R₁ isH, phenyl or C₁₋₄ alkoxy optionally substituted with a carbocycle suchas phenyl. In a particular embodiment R₁ is H. In another particularembodiment R₁ is methoxy, ethoxy, propyloxy, butyloxy, isobutyloxy,s-butyloxy, t-butyloxy, phenoxy or benzyloxy. In yet another particularembodiment R₁ is NH₂. In a particularly preferred embodiment R₁ isethoxy. In another particularly preferred embodiment R₁ is isobutyloxy.In another particularly preferred embodiment R₁ is alkoxy substitutedwith amino, for example 2-aminoethoxy, N-morpholinoethoxy,N,N-dialkyaminoethoxy, quaternary ammonium hydroxy alkoxy (e.g.trimethylammoniumhydroxyethoxy).

R₂₋₅ are independently H, hydroxyl, mercapto, halogen, cyano, amino,amidine, guanidine, nitro or alkoxy; or R₃ and R₄ together form a fusedcarbocycle or heterocycle optionally substituted with hydroxyl, halogen,oxo, thio, amino, amidine, guanidine or alkoxy. In a particularembodiment R₂ and R₃ are independently H, F, Cl, Br or I. In anotherparticular embodiment, R₄ and R₅ are both H. In another particularembodiment, one of R₂ and R₃ is a halogen while the other is hydrogen ora halogen. In a particularly preferred embodiment, R₃ is Cl while R₂, R₄and R₅ are each H. In another particularly preferred embodiment, R₂ andR₃ are both Cl while R₄ and R₅ are both H.

R₆ is H or a hydrocarbon chain optionally substituted with a carbocycleor a heterocycle. In a preferred embodiment, R₆ is H or alkyl i.e.methyl, ethyl, propyl, butyl, i-butyl, s-butyl or t-butyl. In aparticular embodiment R₆ is H.

In a preferred embodiment, compounds of the invention have the generalformula (Ia)-(If)

wherein Cy, Y, L and R₁₋₆ are as previously defined. In a particularlypreferred embodiment, the carbon atom marked with an asterisk (*) incompounds of formula (Ia)-(If) is chiral. In a particular embodiment,the carbon atom has an R-configuration. In another particularembodiment, the carbon atom has an S-configuration.

Particular compounds of the invention include:

and salts, solvates, hydrates and esters thereof.

It will be appreciated that compounds of the invention may incorporatechiral centers and therefore exist as geometric and stereoisomers. Allsuch isomers are contemplated and are within the scope of the inventionwhether in pure isomeric form or in mixtures of such isomers as well asracemates. Stereoisomeric compounds may be separated by establishedtechniques in the art such as chromatography, i.e. chiral HPLC, orcrystallization methods.

“Pharmaceutically acceptable” salts include both acid and base additionsalts. Pharmaceutically acceptable acid addition salt refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like,and organic acids may be selected from aliphatic, cycloaliphatic,aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic classesof organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid,malic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid,anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonicacid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

Pharmaceutically acceptable base addition salts include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

Compounds of the invention may be prepared according to establishedorganic synthesis techniques from starting materials and reagents thatare commercially available or from starting materials that may beprepared from commercially available starting materials. Many standardchemical techniques and procedures are described in March, J., “AdvancedOrganic Chemistry” McGraw-Hill, New York, 1977; and Collman, J.,“Principles and Applications of Organotransition Metal Chemistry”University Science, Mill Valley, 1987; and Larock, R., “ComprehensiveOrganic Transformations” Verlag, N.Y., 1989. It will be appreciated thatdepending on the particular substituents present on the compounds,suitable protection and deprotection procedures will be required inaddition to those steps described herein. Numerous protecting groups aredescribed in Greene and Wuts, Protective Groups in Organic Chemistry, 2dedition, John Wiley and Sons, 1991, as well as detailed protection anddeprotection procedures. For example, suitable amino protecting groupsinclude t-butyloxycarbonyl (Boc), fluorenyl-methyloxycarbonyl (Fmoc),2-trimethylsilyl-ethyoxy-carbonyl (Teoc),1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), allyloxycarbonyl(Alloc), and benzyloxycarbonyl (Cbz). Carboxyl groups can be protectedas fluorenyl-methyl groups, or alkyl esters i.e. methyl or ethyl, oralkenyl esters such as allyl. Hydroxyl groups may be protected withtrityl, monomethoxytrityl, dimethoxy-trityl, and trimethoxytritylgroups.

Compounds may be prepared according to organic synthetic proceduresdescribed in U.S. patent application Ser. No. 09/6446,330 filed on Sep.14, 2000, the entirety of which is incorporated herein by reference.Generally, compounds may be prepared according to reaction scheme 1.

Referring to scheme 1, a commercially available glycine amino acidresidue is protected at the amino (e.g. fmoc) and carboxyl groups (Pr)or else immobilized on a solid support. The amino protecting group isremoved with a suitable reagent and is reacted with diphenylketimine andsubsequently alkylated at the alpha carbon with (iii) halo-X-Cy to giveintermediate (vi). The imine (vi) is converted to the free amine (v) andthen coupled with intermediate (vi) to provide the compound of theinvention which is optionally deprotected at the carboxyl group to givefree acid (vii). The free acid in turn may be esterified or amidatedaccording to the definitions of substituent R₁.

In a particular embodiment, compounds of formula (Ib) of the inventionmay be prepared according to scheme 2.

Referring to scheme 2, starting compound (i), commercially available orsynthesized from commercially available reagents, is reacted withN-hydroxymethylphthalamide to give intermediate (ii) which is reactedwith hydrazine to yield the free amine (iii). The amine is Boc protected(iv) by reacting with Boc₂O and sodiumbicarbonate and then reacted withtriflic anhydride to give intermediate (v). The triflate intermediate(v) is then converted to the methyl ester intermediate (vi) by reactingwith palladium(II) acetate and 1,3-bi(diphenylphosphino propane (dppp)and subsequently with diisopropyl ethylamine (DIPEA). The Boc group of(vi) is removed with TFA and then reacted with carboxylic acid (vii) togive intermediate (viii). In a preferred embodiment of scheme 2,intermediate (vii) Y—L—C(O)OH is furylacrylic acid or thienylacrylicacid. The methyl ester of (viii) is removed with LiOH to give the freeacid which is reacted with the N-Boc protected diaminopropanoicacid/ester (x) to yield intermediate (xi). The Boc group of (xi) isremoved with TFA and then reacted with carboxyl-substituted non-aromaticring (xii) to give final compound (Ib) of the invention.

In another particular embodiment compounds of formula (Ic) of theinvention may be prepared according to scheme 3.

Referring to scheme 3, carboxylate starting reagent (i) is coupled withamine reagent (ii) Y—L—NHR₆ to give intermediate (iii) which is coupledwith (iv) to yield compound of the invention (v). In a preferredembodiment of scheme 3, Y—L— is benzyl, optionally substituted withhydroxy, halogen, alkyl or alkoxy. More preferably Y—L— is3-hydroxy-benzyl.

In another particular embodiment, compounds of formula (Id) of theinvention may be prepared according to scheme 4.

Referring to scheme 4, starting compound (i), prepared according to theprocedures described in scheme 2, is converted to the iodo intermediate(ii) and reacted with alkyne (iii) to give intermediate (iv). Alkyne(iii) is prepared by reacting Y—COOH with Br—C≡CH in THF. Intermediate(iv) is then converted to the alkane (v) by reacting with Rh/Al₂O₃ in H₂atmosphere and the ester group converted to the free acid by reactingwith LiI in pyridine to give (vi). Intermediate (vi) is reacted withamino acid (vii) to give compound of the invention (viii). In aparticular embodiment of scheme 4, Y is phenyl optionally substitutedwith alkyl, hydroxy or halogen. In a particularly preferred embodiment Yis 3-chloro-phenyl or 3-hydroxy-phenyl.

In another particular embodiment, compounds of formula (Ie) of theinvention may be prepared according to scheme 5.

Referring to scheme 5, starting compound (i) is reacted with triflicanhydride and 2,6-lutidine to give intermediate (ii) which is convertedto methyl ester (iii) by reacting with palladium(II)acetate,1,3-bi(diphenylphosphino propane (dppp) and subsequently withdiisopropyl ethylamine (DIPEA) in DMF and methanol. The ester (iii) isthen reacted with CrO₃ in acetic acid and anhydride to give aldehyde(iv) which is reacted with Grignard reagent ethynyl-magnesium bromide inTHF to give alkyne intermediate (v). Iodo reagent (vi) Y—I is reactedwith (v) to give intermediate (vii) which is converted to the alkane(viii) by reacting with Rh/Al₂O₃ under hydrogen atmosphere. The methylester is converted to free acid (ix) with LiI in pyridine which is thencoupled to amino acid residue (x) to give compound of the invention(xi). In preferred embodiments of scheme 5, Y is phenyl, optionallysubstituted with hydroxy, halogen, alkyl or alkoxy. In more preferredembodiments, Y is 3-hydroxy-phenyl or 3-chloro-phenyl.

Compounds of the invention bind to LFA-1 preferentially over Mac-1.Accordingly, in an aspect of the invention, there is provided a methodof inhibiting the binding of LFA-1 to ICAMs (cellular adhesionmolecules), the method comprising contacting LFA-1 with a compound offormula (I). The method may be carried out in vivo or ex vivo as asolution based or cell based assay wherein the compound of the inventionis introduced to LFA-1 in the presence of a putative or known ligand(such as ICAM-1). The compound of the invention may be labeled, forexample isotopically radiolabeled, or labeled with a fluorophore such asfluorescein isothiocyanate (FITC), to facilitate detection of ligandbinding or reduction thereof to the protease. Thus compounds of theinvention are useful for diagnostic and screening assays.

Compounds of the invention are therapeutically and/or prophylacticallyuseful for treating diseases or conditions mediated by LFA-1 activity.Accordingly in an aspect of the invention, there is provided a method oftreating a disease or condition mediated by LFA-1 in a mammal, i.e. ahuman, comprising administering to said mammal an effective amount of acompound of the invention. By “effective amount” is meant an amount ofcompound which upon administration is capable of reducing the activityof LFA-1; or the amount of compound required to prevent, inhibit orreduce the severity of any symptom associated with an LFA-1 mediatedcondition or disease upon administration.

Compounds of the invention or compositions thereof are useful intreating conditions or diseases including: psoriasis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis), dermatitis, meningitis, encephalitis, uveitis,allergic conditions such as eczema and asthma, conditions involvinginfiltration of T-cells and chronic inflammatory responses, skinhypersensitivity reactions (including poison ivy and poison oak);atherosclerosis, autoimmune diseases such as rheumatoid arthritis,systemic lupus erythematosis (SLE), diabetes mellitus, multiplesclerosis, Reynaud's syndrome, autoimmune thyroiditis, experimentalautoimmune encephalomyelitis, Sjorgen's syndrome, juvenile onsetdiabetes, and immune responses associated with delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia; diseases involving leukocyte diapedesis; CNS inflammatorydisorder, multiple organ injury syndrome secondary to septicaemia ortrauma; autoimmune hemolytic anemia; myasthemia gravis; antigen-antibodycomplex mediated diseases; all types of transplantations, includinggraft vs. host or host vs. graft disease, HIV and rhinovirus infection,pulmonary fibrosis, alopecia, scleredoma, endometriosus, vitiligo,ischemic reperfusion injury mediated by neutrophils such as acutemyocardial infarction, restenosis following PTCA, invasive proceduressuch as cardiopulmonary bypass surgery, cerebral edema, stroke,traumatic brain injury, hemorragic shock, burns, ischemic kidneydisease, multi-organ failure, wound healing and scar formation,atherosclerosis.

The actual amount of compound administered and the route ofadministration will depend upon the particular disease or condition aswell as other factors such as the size, age, sex and ethnic origin ofthe individual being treated and is determined by routine analysis. Ingeneral, intravenous doses will be in the range from about 0.01-1000mg/kg of patient body weight per day, preferably 0.1 to 20 mg/kg andmore preferably 0.3 to 15 mg/kg. Administration may be once or multipletimes per day for several days, weeks or years or may be a few times perweek for several weeks or years. The amount of compound administered byother routes will be that which provides a similar amount of compound inplasma compared to the intravenous amounts described which will takeinto consideration the plasma bioavailability of the particular compoundadministered.

In methods of the invention, the compound may be administered orally(including buccal, sublingual, inhalation), nasally, rectally,vaginally, intravenously (including intra-arterially), intradermally,subcutaneously, intramuscularly and topically. Compounds will beformulated into compositions suitable for administration for examplewith carriers, diluents, thickeners, adjuvants etc. as are routine inthe formulation art. Accordingly, another aspect of the inventionprovides pharmaceutical compositions comprising a compound of formula(I) and a pharmaceutically acceptable carrier, excipient or adjuvant andmay also include additional active ingredients such asanti-inflammatories e.g. NSAIDs.

Dosage forms include solutions, powders, tablets, capsules, gelcapsules, suppositories, topical ointments and creams and aerosols forinhalation. Formulations for non-parenteral administration may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives. Pharmaceutically acceptable organic orinorganic carrier substances suitable for non-parenteral administrationwhich do not deleteriously react with compounds of the invention can beused. Suitable pharmaceutically acceptable carriers include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike. The formulations can be sterilized and, if desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings flavorings and/or aromatic substances and the like which donot deleteriously react with compounds of the invention. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

Compounds of the invention exhibit high oral bioavailability.Accordingly, in a preferred embodiment, compounds of the invention areadministered via oral delivery. Compositions for oral administrationinclude powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, troches, tablets or SECs (softelastic capsules or caplets). Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids, carrier substances or binders may bedesirably added to such formulations. Such formulations may be used toeffect delivering the compounds to the alimentary canal for exposure tothe mucosa thereof. Accordingly, the formulation can consist of materialeffective in protecting the compound from pH extremes of the stomach, orin releasing the compound over time, to optimize the delivery thereof toa particular mucosal site. Enteric coatings for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol and sorbitan monoleate.

Various methods for producing formulations for alimentary delivery arewell known in the art. See, generally Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990. The formulations of the invention can be converted in a knownmanner into the customary formulations, such as tablets, coated tablets,pills, granules, aerosols, syrups, emulsions, suspensions and solutions,using inert, non-toxic, pharmaceutically suitable excipients orsolvents. The therapeutically active compound should in each case bepresent in a concentration of about 0.1% to about 99% by weight of thetotal mixture, that is to say in amounts which are sufficient to achievethe desired dosage range. The formulations are prepared, for example, byextending the active compounds with solvents and/or excipients, ifappropriate using emulsifying agents and/or dispersing agents, and, forexample, in the case where water is used as the diluent, organicsolvents can be used as auxiliary solvents if appropriate.

Compositions may also be formulated with binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrates (e.g., starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulfate). Tablets may be coated bymethods well known in the art. The preparations may also containflavoring, coloring and/or sweetening agents as appropriate.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing predetermined amounts of the active ingredients; aspowders or granules; as solutions or suspensions in an aqueous liquid ora non-aqueous liquid; or as oil-in-water emulsions or water-in-oilliquid emulsions. A tablet may be made by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein.

EXAMPLES

Abbreviations used in the following section: Boc=t-butyloxycarbonyl;Boc₂O=t-butyloxycarbonyl anhydride; DMA=dimethylacetimide;DMF=dimethylformamide; Hobt=1-hydroxybenztriazole; TFA=trifluoroaceticacid; DCM=dichloromethane; MeOH=methanol; HOAc=acetic acid;HCl=hydrochloric acid; H₂SO₄=sulfuric acid; K₂CO₃=potassium carbonate;THF=tetrahydrofuran; EtOAc=ethyl acetate; DIPEA=diisopropylethylamine;NaHCO₃=sodium bicarbonate; ACN=acetonitrile;Na₂.EDTA=ethylenediaminetetraacetic acid sodium salt; TBAF=tetrabutylammonium fluoride; EDC=1(3-dimethylaminopropyl)-3-ethylcarbodiimide.HCl;TEA=triethylamine; MgSO₄=magnesium sulfate; TES=triethylsilane;Et₂O=diethyl ether; BBr₃=boron tribromide

Example 1 Synthesis of Compounds 16, 17, 38-40, 46-50

A round bottom flask was equipped with an efficient overhead stirrer andcharged with concentrated H₂SO₄ (2.7×volume of H₂O) and H₂O and cooledto ˜−5° C. with an ethanol/ice bath. Once cool, 1 equivalent 2.6dichloro phenol and 1 equivalent of N-(hydroxymethyl)phthalimide wereadded with vigorous stirring. The reaction was kept cool for 4 hours andthen allowed to warm to room temperature overnight with constantstirring. The reaction generally proceeded to a point where there wasjust a solid in the round bottom flask. At that point EtOAc and H₂O wereadded and stirred into the solid. Large chunks were broken up and thenthe precipitate was filtered and washed with more EtOAc and H₂O. Theproduct was then used without further purification after dryingovernight under vacuum.

1 equivalent of the dry product and methanol (22.5 ml×#g of startingmaterial) was added to a round bottom flask equipped with a H₂Ocondenser and stirring bar. 1.2 equivalents of hydrazine mono hydratewas added and the mixture refluxed for 4 hours. After cooling to roomtemperature, concentrated HCl (4.5 ml×#g of starting material) wascarefully added. Upon completion of the addition, the mixture wasrefluxed overnight (>8 hours).

The reaction was cooled to 0° C. and the precipitated by-product wasremoved by filtration. The filtrate was then concentrated in vacuo.

The crude amine residue was dissolved in a 3:2 THF/H₂O solution. 1.1equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were added andthe mixture was stirred overnight. The reaction was concentrated, andthe residue was partitioned between H₂O and Et₂O. The aqueous layer wasextracted with Et₂O and the combined organic layers were dried overMgSO₄ and concentrated in vacuo to a solid. Recrystallization from hotmethanol and H₂O provided pure product.

1 equivalent of the Boc protected amine and 1.5 equivalents of2,6-lutidine was dissolved, with mild heating when necessary, in DCM ina round bottom flask. Once the starting material had completelydissolved, the mixture was cooled to −78° C. under N₂ with a dry iceethanol bath. Once cool, 2.5 equivalents of triflic anhydride was addedand the reaction was allowed to slowly come to room temperature withstirring. The reaction was monitored by TLC and was generally done in 4hours. Upon completion, the reaction was concentrated in vacuo and theresidue partitioned between EtOAc and H₂O. The organic layer was washedtwice with 0.1N H₂SO₄, twice with saturated NaHCO₃, once with brine,dried over MgSO₄ and concentrated in vacuo. The residue was thenpurified on silica gel using DCM as eluent to provide pure triflate.

1 equivalent of triflate was dissolved in DMF and MeOH in the glassinsert of a high pressure Parr bomb. The starting material was thendegassed while stirring with CO for 10 minutes. 0.15 equivalentspalladium(II) acetate and 0.15 equivalents of 1,3-bis(diphenylphosphino)propane were then added and the mixture was then degassed while stirringwith CO for another 10 minutes at which time 2.5 equivalents ofdiisopropyl ethyl amine was added. After properly assembling the bomb,it was charged with 300 psi CO gas and heated to 70° C. with stirringovernight. The bomb was then cooled and vented. The mixture wastransferred to a round bottom flask and concentrated in vacuo. Theresidue was then purified on silica gel using DCM with 1% acetone and 1%TEA as eluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. The TFAsalt of the amine was dissolved in Et₂O and washed twice with a 10%solution of K₂CO₃ in H₂O and once with brine. The organic layer was thendried over MgSO₄, filtered and concentrated in vacuo.

1 equivalent of the free based amine, 3 equivalents of furylacrylicacid, 3 equivalents of EDC and 1 equivalent of Hobt were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents of the appropriate commerciallyavailable carboxylic acid (compound 16, N-acetyl-D-proline; compound 17,N-acetyl-L-proline; compound 38, (−)-2-oxo-4-thiazolidinecarboxylicacid; compound 39, 1-cyclohexene-1-carboxylic acid; compound 40,(4R)-(−)-2-thioxo-4-thiazolidinecarboxylic acid; compound 45,cyclobutanecarboxylic acid; compound 46, cyclopentane-carboxylic acid;compound 47, cyclohexanecarboxylic acid; compound 48,3,4-dihydro-2,2-dimethyl-4-oxo-2H-pyran-6-carboxylic acid; compound 49,ethyl 1,3-dithiolane-2-carboxylate (2 equivalents of the ethyl ester wassaponified with 3 equivalents of LiOH.H₂O in THF/H₂O (3/1) The reactionwas monitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was used without further purification); compound 50,cyclopropanecarboxylic acid; compound 51, tetrahydro-2-furoic acid), 2equivalents of EDC, 1 equivalent of Hobt and 3 equivalents of DIPEA weredissolved DMA. The reaction was stirred at room temperature andmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasconcentrated in vacuo. The resulting oil was re suspended in Et₂O andwashed twice with 0.1 N H₂SO₄, twice with saturated NaHCO₃, and oncewith brine. The organic layer was then dried over MgSO₄, filtered andconcentrated in vacuo. The residue was then purified on silica get using5% methanol in DCM as eluent to provide pure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was resuspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The resulting acid was then purifiedby reverse phase HPLC, verified by electrospray mass spectrometry andlyophilized to a powder.

Example 2 Synthesis of Compounds 1-15,41,43

A round bottom flask was equipped with an efficient overhead stirrer andcharged with concentrated H₂SO₄ (2.7×volume of H₂O) and H₂O and cooledto ˜−5° C. with an ethanol/ice bath. Once cool, 1 equivalent 2.6dichloro phenol and 1 equivalent of N-(hydroxymethyl)phthalimide wereadded with vigorous stirring. The reaction was kept cool for 4 hours andthen allowed to warm to room temperature overnight with constantstirring. The reaction generally proceeds to a point where there wasjust a solid in the round bottom flask. At this point EtOAc and H₂O wereadded and stirred into the solid. Large chunks were broken up and thenthe precipitate was filtered and washed with more EtOAc and H₂O. Theproduct was then used without further purification after dryingovernight under vacuum.

1 equivalent of the dry product and methanol (22.5 ml×#g of startingmaterial) was added to a round bottom flask equipped with a H₂Ocondenser and stirring bar. 1.2 equivalents of hydrazine mono hydratewas added and the mixture refluxed for 4 hours. After cooling to roomtemperature, concentrated HCl (4.5 ml×#g of starting material) wascarefully added. Upon completion of the addition, the mixture wasrefluxed overnight (>8 hours). The reaction was cooled to 0° C. and theprecipitated by-product was removed by filtration. The filtrate was thenconcentrated in vacuo.

The crude amine residue was dissolved in a 3:2 THF/H₂O solution. 1.1equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were added andthe mixture was stirred overnight. The reaction was concentrated, andthe residue was partitioned between H₂O and Et₂O. The aqueous layer wasextracted with Et₂O and the combined organic layers were dried overMgSO₄ and concentrated in vacuo to a solid. Recrystallization from hotmethanol and H₂O provided pure product.

1 equivalent of the Boc protected amine and 1.5 equivalents of2,6-lutidine was dissolved, with mild heating when necessary, in DCM ina round bottom flask. Once the starting material had completelydissolved, the mixture was cooled to −78° C. under N₂ with a dry iceethanol bath. Once cool, 2.5 equivalents of triflic anhydride was addedand the reaction was allowed to slowly come to room temperature withstirring. The reaction was monitored by TLC and was generally done in 4hours. Upon completion, the reaction was concentrated in vacuo and theresidue partitioned between EtOAc and H₂O. The organic layer was washedtwice with 0.1N H₂SO₄, twice with saturated NaHCO₃, once with brine,dried over MgSO₄ and concentrated in vacuo. The residue was thenpurified on silica gel using DCM as eluent to provide pure triflate.

1 equivalent of triflate was dissolved in DMF and MeOH in the glassinsert of a high pressure Parr bomb. The starting material was thendegassed while stirring with CO for 10 minutes. 0.15 equivalentspalladium(TI) acetate and 0.15 equivalents of 1,3-bis(diphenylphosphino)propane were then added and the mixture was then degassed while stirringwith CO for another 10 minutes at which time 2.5 equivalents ofdiisopropyl ethyl amine was added. After properly assembling the bomb,it was charged with 300 psi CO gas and heated to 70° C. with stirringovernight. The bomb was then cooled and vented. The mixture wastransferred to a round bottom flask and concentrated in vacuo. Theresidue was then purified on silica gel using DCM with 1% acetone and 1%TEA as eluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. The TFAsalt of the amine was dissolved in Et₂O and washed twice with a 10%solution of K₂CO₃ in H₂O and once with brine. The organic layer was thendried over MgSO₄, filtered and concentrated in vacuo.

1 equivalent of the free based amine, 3 equivalents of furylacrylicacid, 3 equivalents of EDC and 1 equivalent of Hobt were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents of the appropriate commerciallyavailable carboxylic acid ((N-Boc acids were purchased where available.Other acids were purchased as the free amine and Boc protected by thefollowing procedure: The amine was dissolved in a 3:2 THF/H₂O solution.1.1 equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were addedand the mixture was stirred overnight. The reaction was concentrated toremove the THF, and the resulting aqueous layer was partitioned withhexanes. The aqueous layer was then acidified to pH 2 with 1N HCl andthen partitioned twice with EtOAc. The combined organic layers weredried over MgSO₄ and concentrated in vacuo. The resulting product wasused without further purification) compound 1 D,L-pipecolinic acid;compound 2, nipecotic acid; compound 3, isonipecotic acid; compound 4,N-Boc-L-proline; compound 5, N-Boc-D-proline; compound 6,Boc-L-thiazolidine-4-carboxylic acid; compound 7, N-Boc-L-pyroglutamicacid; compound 8, N-Boc-D-pyroglutamic acid; compound 9, L-pipecolinicacid; compound 10, D-cis-4-hydroxyproline; compound 11,L-cis-4-hydroxyproline; compound 12, D-hydroxyproline; compound 13, (2S,3S)-3-methylpyrrolidine-2-carboxylic acid; compound 14,N-Boc-L-hydroxyproline; compound 15, Boc-D-thiazolidine-4-carboxylicacid; compound 41, L-3-hydroxyproline; compound 43,trans-3-azabicyclo[3.1.0]-hexane-2-carboxylic acid), 2 equivalents ofEDC, 1 equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo.

Where appropriate the Boc protected residue was dissolved in a solutionof TFA in DCM (1:1). After 20 minutes, the reaction was concentrated invacuo. The resulting oil was dissolved in toluene and thenreconcentrated in vacuo. The resulting acid was then purified by reversephase HPLC, verified by electrospray mass spectrometry and lyophilizedto a powder.

Example 3 Synthesis of Compounds 18-21

1 equivalent of 4-amino-2,6-dichlorophenol was dissolved in a 3:2THF/H₂O solution. 1.1 equivalents of solid NaHCO₃ and 1.1 equivalents ofBoc₂O were added and the solution was stirred overnight. The reactionwas concentrated, and the residue was partitioned between H₂O and Et₂O.The aqueous layer was extracted with Et₂O and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo to a solid.Recrystallization out of Et₂O/hexane provided pure product.

1 equivalent of the phenol was dissolved in DCM containing 2.6equivalents of 2,6-lutidine and the mixture was cooled to −78° C. Afteradding 1.25 equivalents of triflic anhydride the stirring reaction wasallowed to warm to room temperature overnight. The reaction was thenconcentrated, and the residue was partitioned between Et₂O and H₂O. Theaqueous layer was extracted with Et₂O and the combined organic layerswere dried over MgSO₄ and concentrated in vacuo. The residue waspurified by silica gel flash chromatography (9:1 hexane/Et₂O) to providethe pure triflate.

To a stirring solution of 1 equivalent of the triflate in a 2/1 mixtureof DMF/MeOH was added 0.15 equivalents of1,3-bis(diphenylphosphino)-propane and 2.5 equivalents of TEA. Carbonmonoxide gas was bubbled through this solution for 15 minutes, then 0.15equivalents of Pd(OAc)2 was added and the reaction was stirred at 70° C.for 5-7 hours under an atmosphere of CO (using a balloon filled withCO). The reaction was then concentrated in vacuo, and the residue waspartitioned between Et₂O and H₂O. The aqueous layer was extracted twicewith Et₂O and the combined organic layers were dried over MgSO₄,filtered through a plug of silica gel and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (9:1:0.02hexane/DCM/Et₂O) to provide the pure methyl ester.

1 equivalent of the Boc-aniline was dissolved in methanol and thesolution saturated with HCl. The reaction was heated at 50° C. for 3 h,then concentrated in vacuo. The pale yellow solid was heated in 35%H₂SO₄ until complete dissolution occurred. Upon cooling the mixture bythe addition of ice H₂O the amine bisulfate precipitated. The reactionflask was cooled in an ice bath and the mixture stirred vigorously while1.1 equivalents of sodium nitrite in H₂O was added drop wise. Thereaction was stirred at 0° C. for another 1.5 hours. An aqueous solutionof 10 equivalents of KI was added, followed immediately with 17equivalents CuI. The reaction was stirred at room temperature for 14hours, then extracted 3 times with Et₂O. The combined organic layerswere washed with 1M NaHCO₃, brine, and dried over MgSO₄, thenconcentrated in vacuo. The residue was purified by silica gel flashchromatography (95:5 hexane/Et₂O) to provide the pure aryl iodide methylester.

A solution of 1 equivalent of 3-Chlorobenzaldehyde in THF was cooled to−78° C. and 1.1 equivalents of 0.5M ethynylmagnesium bromide/THF wasadded. After stirring the reaction at room temperature for 3 hours, itwas diluted with Et₂O and washed twice with 10% citric acid. Thecombined aqueous layers were back-extracted once with Et₂O. The combinedorganic layers were washed twice with saturated aqueous NaHCO₃, driedover MgSO₄ and concentrated in vacuo. The residue was purified by silicagel flash chromatography (4:1 to 3:2 hexane/Et₂O) to provide the purealkyne.

1 equivalent of the aryl iodide methyl ester was dissolved in EtOAc andthe solution was degassed by passing N2 through a pipette and into thesolution for 10 minutes. 1.25 equivalents of the alkyne was added,followed by 0.02 equivalents ofdichlorobis(triphenylphosphine)palladium(II), 0.04 equivalents of CuIand 5 equivalents TEA. The reaction was stirred for 14 hours, dilutedwith EtOAc, washed twice with 5% Na₂.EDTA, brine and then dried overMgSO₄ and concentrated in vacuo. The residue was purified by silica gelflash chromatography (gradient elution, using Et₂O to EtOAc) to providethe pure aryl alkyne.

1 equivalent of the aryl alkyne was dissolved in MeOH and the solutionwas degassed by passing N2 through a pipette and into the solution for10 minutes. The 5% Rh/Al₂O₃ was added, one balloon-full of hydrogen waspassed through the solution, and the reaction was stirred under anatmosphere of H₂ (using a balloon) for 7 hours, after which the reactionwas filtered through a pad of celite and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (gradientelution, using Et₂O to EtOAc) to provide the pure product.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents of the appropriate commerciallyavailable carboxylic acid ((N-Boc acids were purchased where available.Other acids were purchased as the free amine and Boc protected by thefollowing procedure: The amine was dissolved in a 3:2 THF/H₂O solution.1.1 equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were addedand the mixture was stirred overnight. The reaction was concentrated toremove the THF, and the resulting aqueous layer was partitioned withhexanes. The aqueous layer was then acidified to pH 2 with 1N HCl andthen partitioned twice with EtOAc. The combined organic layers weredried over MgSO₄ and concentrated in vacuo. The resulting product wasused without further purification) example 18, N-Boc-D-proline; example19, N-Boc-L-proline; example 20, Boc-L-thiazolidine-4-carboxylic acid;example 21, isonipecotic acid; 2 equivalents of EDC, 1 equivalent ofHobt and 3 equivalents of DIPEA were dissolved DMA. The reaction wasstirred at room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was resuspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo.

The Boc protected residue was dissolved in a solution of TFA in DCM(1:1). After 20 minutes, the reaction was concentrated in vacuo. Theresulting oil was dissolved in toluene and then reconcentrated in vacuo.The resulting acid was then purified by reverse phase HPLC, verified byelectrospray mass spectrometry and lyophilized to a powder.

Example 4 Synthesis of Compounds 22-25

1 equivalent of 4-amino-2,6-dichlorophenol was dissolved in a 3:2THF/H₂O solution. 1.1 equivalents of solid NaHCO₂ and 1.1 equivalents ofBoC₂O were added and the solution was stirred overnight. The reactionwas concentrated, and the residue was partitioned between H₂O and Et₂O.The aqueous layer was extracted with Et₂O and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo to a solid.Recrystallization out of Et₂O/hexane provided pure product.

1 equivalent of the phenol was dissolved in DCM containing 2.6equivalents of 2,6-lutidine and the mixture was cooled to −78° C. Afteradding 1.25 equivalents of triflic anhydride the stirring reaction wasallowed to warm to room temperature overnight. The reaction was thenconcentrated, and the residue was partitioned between Et₂O and H₂O. Theaqueous layer was extracted with Et₃O and the combined organic layerswere dried over MgSO₄ and concentrated in vacuo. The residue waspurified by silica gel flash chromatography (9:1 hexane/Et₂O) to providethe pure triflate.

To a stirring solution of 1 equivalent of the triflate in a 2/1 mixtureof DMF/MeOH was added 0.15 equivalents of1,3-bis(diphenylphosphino)-propane and 2.5 equivalents of TEA. Carbonmonoxide gas was bubbled through this solution for 15 minutes, then 0.15equivalents of Pd(OAc)2 was added and the reaction was stirred at 70° C.for 5-7 hours under an atmosphere of CO (using a balloon filled withCO). The reaction was then concentrated in vacuo, and the residue waspartitioned between Et₂O and H₂O. The aqueous layer was extracted twicewith Et₂O and the combined organic layers were dried over MgSO₄,filtered through a plug of silica gel and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (9:1:0.02hexane/DCM/Et₂O) to provide the pure methyl ester.

1 equivalent of the Boc-aniline was dissolved in methanol and thesolution saturated with HCl. The reaction was heated at 50° C. for 3 h,then concentrated in vacuo. The pale yellow solid was heated in 35%H₂SO₄ until complete dissolution occurred. Upon cooling the mixture bythe addition of ice H₂O the amine bisulfate precipitated. The reactionflask was cooled in an ice bath and the mixture stirred vigorously while1.1 equivalents of sodium nitrite in H₂O was added drop wise. Thereaction was stirred at 0° C. for another 1.5 hours. An aqueous solutionof 10 equivalents of KI was added, followed immediately with 17equivalents CuI. The reaction was stirred at room temperature for 14hours, then extracted 3 times with Et₂O. The combined organic layerswere washed with 1M NaHCO₃, brine, and dried over MgSO₄, thenconcentrated in vacuo. The residue was purified by silica gel flashchromatography (95:5 hexane/Et₂O) to provide the pure aryl iodide methylester.

1.3 equivalents of DIPEA was added to a heterogeneous mixture of 1equivalent of 3-hydroxybenzoic acid, 1.3 equivalents of N,O-dimethylhydroxylamine hydrochloride, 1.3 equivalents of HOBt and 1.3equivalents of EDC stirring in DMF. All solids eventually dissolved asthe mixture was stirred at room temperature for 28 hours. Afterconcentrating the mixture, the residue was partitioned between Et₂O andH₂O. The aqueous layer was extracted three times with Et₂O and thecombined organic layers were dried over MgSO₄, and concentrated invacuo. The residue was purified by silica gel flash chromatography(Et₂O) to provide the pure hydroxamate.

1 equivalent of the hydroxamate, 2.2 equivalents oft-butyldimethyl silylchloride and 3 equivalents of imidizole were dissolved in DMF andstirred at room temperature. The reaction was monitored by TLC (9/1DCM/MeOH). Upon reaction completion, the mixture was concentrated invacuo. The resulting oil was re suspended in Et₂O and washed twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The product was thenused with out further purification.

To a stirred −78° C. solution of 1 equivalent of the protectedhydroxamate in THF was added a solution of 1.2 equivalents of 1.5 MDIBAL in toluene drop wise. The reaction mixture was stirred for anadditional 3 hours at −78° C. or until TLC showed clean formation ofproduct, with only a trace of starting material. The reaction wasquenched by adding to a separatory funnel containing Et₂O and 0.35MNaHSO₄. The layers were separated. The aqueous layer was extracted threetimes with ethyl ether. The combined organic layers were washed twicewith 1N HCl, saturated aqueous NaHCO₃, and over MgSO₄, filtered througha plug of silica gel, and concentrated in vacuo. No further purificationof the aldehyde was necessary.

A solution of 1 equivalent of the protected aldehyde in THF was cooledto −78° C. and 1.1 equivalents of 0.5M ethynylmagnesium bromide/THF wasadded. After stirring the reaction at room temperature for 3 hours, itwas diluted with Et₂O and washed twice with 10% citric acid. Thecombined aqueous layers were back-extracted once with Et₂O. The combinedorganic layers were washed twice with saturated aqueous NaHCO₃, driedover MgSO₄ and concentrated in vacuo. The residue was purified by silicagel flash chromatography (4:1 to 3:2 hexane/Et₂O) to provide the purealkyne.

1 equivalent of the aryl iodide methyl ester was dissolved in EtOAc andthe solution was degassed by passing N2 through a pipette and into thesolution for 10 minutes. 1.25 equivalents of the alkyne was added,followed by 0.02 equivalents ofdichlorobis(triphenylphosphine)palladium(II), 0.04 equivalents of CuIand 5 equivalents TEA. The reaction was stirred for 14 hours, dilutedwith EtOAc, washed twice with 5% Na₂.EDTA, brine and then dried overMgSO₄ and concentrated in vacuo. The residue was purified by silica gelflash chromatography (gradient elution, using Et₂O to EtOAc) to providethe pure aryl alkyne.

1 equivalent of the aryl alkyne was dissolved in MeOH and the solutionwas degassed by passing N2 through a pipette and into the solution for10 minutes. The 5% Rh/Al₂O₃ was added, one balloon-full of hydrogen waspassed through the solution, and the reaction was stirred under anatmosphere of H₂ (using a balloon) for 7 hours, after which the reactionwas filtered through a pad of celite and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (gradientelution, using Et₂O to EtOAc) to provide the pure product.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents of the appropriate commerciallyavailable carboxylic acid ((N-Boc acids were purchased where available.Other acids were purchased as the free amine and Boc protected by thefollowing procedure: The amine was dissolved in a 3:2 THF/H₂O solution.1.1 equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were addedand the mixture was stirred overnight. The reaction was concentrated toremove the THF, and the resulting aqueous layer was partitioned withhexanes. The aqueous layer was then acidified to pH 2 with 1N HCl andthen partitioned twice with EtOAc. The combined organic layers weredried over MgSO₄ and concentrated in vacuo. The resulting product wasused without further purification) example 22, N-Boc-L-proline; example23, N-Boc-D-proline; example 24, Boc-L-thiazolidine-4-carboxylic acid;example 25, D-hydroxy proline; 2 equivalents of EDC, 1 equivalent ofHobt and 3 equivalents of DIPEA were dissolved DMA. The reaction wasstirred at room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The Boc, silyl residue was dissolvedin a solution of TFA in DCM (1:1) with 3 equivalents of TBAF. After 20minutes, the reaction was concentrated in vacuo. The resulting oil wasdissolved in toluene and then reconcentrated in vacuo. The resultingacid was then purified by reverse phase HPLC, verified by electrospraymass spectrometry and lyophilized to a powder.

Example 5 Synthesis of Compounds 26-28, 31

1 equivalent of dimethyl 2-chloroterephthalic acid was dissolved in DCMand cooled to −5° C. in an ice/acetone bath under nitrogen. 1 equivalentof BBr₃ was added drop wise as a solution in DCM over 30 minutes. Thereaction was warmed to room temperature and stirred until complete byTLC (DCM/2% HOAc/2% MeOH). The solution was poured onto ice, and the icewas allowed to melt. The mixture was then partitioned with EtOAc andconcentrated in vacuo. This product was dissolved in H₂O with theaddition of saturated NaHCO₃ until the pH remained above 8. Thissolution was partitioned one time with and equal volume of DCM to removeunreacted diester. The basic solution was acidified at 0° C. withconcentrated HCl to pH=1-1.5, and precipitate was extracted twice withequal volumes of EtOAc. The organics were partitioned once with brineand dried over MgSO₄, filtered and concentrated in vacuo. Product was7:1 of the correct regioisomer by HPLC.

The monoester was dissolved in DCM and transferred to a pre-weighed Parrflask containing a stirring bar. The flask was cooled to −5° C. with adry ice/alcohol bath under nitrogen. Once cool, ˜30 equivalents ofisobutylene was pumped into solution with stirring. 2.1 equivalents ofconcentrated sulfuric acid was added and the flask was sealed with awired rubber stopper and allowed to warm to room temperature withstirring. The solution was stirred until clarification (1-2 days). Oncethe solution was clear, it was cooled to 0° C. in an ice bath. Thestopper was removed and the excess isobutylene was blown off withnitrogen bubbling. Saturated NaHCO₃ was added to neutralize the acid andthe mixture was concentrated in vacuo until no DCM remained. Thesolution was then partitioned into EtOAc. The organics were partitionedtwice with dilute HCl, twice with saturated NaHCO₃, once with brine,dried over MgSO₄, filtered and concentrated in vacuo. The resultingproduct was used with no further purification.

1 equivalent of the methyl ester was dissolved in THF/H₂O (3/1) and 3equivalents of LiOH.H₂O was added. The reaction was monitored by TLC(9/1 DCM/MeOH). Upon completion, the mixture was acidified carefully topH 2 with concentrated HCl and then concentrated in vacuo to remove theTHF. The resulting aqueous layer was washed twice with Et₂O and thecombined organic layers were washed once with brine. The organic layerwas then dried over MgSO₄, filtered and concentrated in vacuo. Thebenzoic acid t-butyl ester was used without further purification.

1 equivalent of 3-methoxybenzonitrile was placed in a Parr bottle withEtOH, 0.02 equivalents of HCl and 10% (w/w) of 10% Pd on carbon. Thevessel was placed in the Parr shaker, charged with 50 psi H2, and shakenfor 12 hours. The reaction filtered through a pad of celite and diluted1:10 with Et₂O. Upon standing over night, fine white needles form. Theproduct was filtered, washed with Et₂O and dried in vacuo. The resultingamine hydrochloride salt was then used with out further purification.

3 equivalents of the benzoic acid t-butyl ester was coupled to 1equivalent of the amine hydrochloride salt using 3 equivalents EDC, 1equivalent of Hobt and 3 equivalents of DIPEA in DMA. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasconcentrated in vacuo. The resulting oil was re suspended in Et₂O andwashed twice with 0.1 N H₂SO₄, twice with saturated NaHCO₃, and oncewith brine. The organic layer was then dried over MgSO₄, filtered andconcentrated in vacuo. The product was then purified on silica get using5% methanol in DCM as eluent to provide pure t-butyl ester.

The t-butyl ester was dissolved in a solution of TFA in DCM (1:1). After20 minutes, the reaction was concentrated in vacuo. The resulting oilwas dissolved in toluene and then concentrated in vacuo twice.

The resulting compound was dissolved in DCM and cooled to −5° C. in anice/acetone bath under nitrogen. 2 equivalents of BBr₃ were added dropwise as a solution in DCM over 30 minutes. The reaction was warmed toroom temperature and stirred until complete by TLC (DCM/2% HOAc/2%MeOH). The solution was poured onto ice, and the ice was allowed tomelt. The mixture was then partitioned twice with EtOAc and the combinedorganic layers were dried over MgSO₄. The filtrate was then passed overa plug of silica gel and concentrated in vacuo to afford pure benzoicacid.

1 equivalent of the benzoic acid, 2 equivalents of commerciallyavailable β-Boc-diaminopropionic acid methyl ester, 2 equivalents ofEDC, 1 equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then re concentrated in vacuo. 1equivalent of this amine, 2 equivalents of the appropriate commerciallyavailable carboxylic acid ((N-Boc acids were purchased where available.Other acids were purchased as the free amine and Boc protected by thefollowing procedure: The amine was dissolved in a 3:2 THF/H₂O solution.1.1 equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were addedand the mixture was stirred overnight. The reaction was concentrated toremove the THF, and the resulting aqueous layer was partitioned withhexanes. The aqueous layer was then acidified to pH 2 with 1N HCl andthen partitioned twice with EtOAc. The combined organic layers weredried over MgSO₄ and concentrated in vacuo. The resulting product wasused without further purification) example 26, cyclohexanecarboxylicacid; example 27, isonipecotic acid; example 28, D,L-pipecolinic acid;example 31, nipecotic acid; 2 equivalents of EDC, 1 equivalent of Hobtand 3 equivalents of DIPEA were dissolved DMA. The reaction was stirredat room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo.

Where appropriate the Boc protected residue was dissolved in a solutionof TFA in DCM (1:1). After 20 minutes, the reaction was concentrated invacuo. The resulting oil was dissolved in toluene and then reconcentrated in vacuo. The resulting acid was then purified by reversephase HPLC, verified by electrospray mass spectrometry and lyophilizedto a powder.

Example 6 Synthesis of Compounds 29, 30

1 equivalent of dimethyl 2-chloroterephthalic acid was dissolved in DCMand cooled to −5° C. in an ice/acetone bath under nitrogen. 1 equivalentof BBr₃ was added drop wise as a solution in DCM over 30 minutes. Thereaction was warmed to room temperature and stirred until complete byTLC (DCM/2% HOAc/2% MeOH). The solution was poured onto ice, and the icewas allowed to melt. The mixture was then partitioned with EtOAc andconcentrated in vacuo. This product was dissolved in H₂O with theaddition of saturated NaHCO₃ until the pH remained above 8. Thissolution was partitioned one time with and equal volume of DCM to removeunreacted diester. The basic solution was acidified at 0° C. withconcentrated HCl to pH=1-1.5, and precipitate was extracted twice withequal volumes of EtOAc. The oraganics were partitioned once with brineand dried over MgSO₄, filtered and concentrated in vacuo. Product was7:1 of the correct regioisomer by HPLC.

The monoester was dissolved in DCM and transferred to a pre-weighed Parrflask containing a stirring bar. The flask was cooled to −5° C. with adry ice/alcohol bath under nitrogen. Once cool, ˜30 equivalents ofisobutylene was pumped into solution with stirring. 2.1 equivalents ofconcentrated sulfuric acid was added and the flask was sealed with awired rubber stopper and allowed to warm to room temperature withstirring. The solution was stirred until clarification (1-2 days). Oncethe solution was clear, it was cooled to 0° C. in an ice bath. Thestopper was removed and the excess isobutylene was blown off withnitrogen bubbling. Saturated NaHCO₃ was added to neutralize the acid andthe mixture was concentrated in vacuo until no DCM remained. Thesolution was then partitioned into EtOAc. The oraganics were partitionedtwice with dilute HCl, twice with saturated NaHCO₃, once with brine,dried over MgSO₄, filtered and concentrated in vacuo. The resultingproduct was used with no further purification.

1 equivalent of the methyl ester was dissolved in THF/H₂O (3/1) and 3equivalents of LiOH.H₂O were added. The reaction was monitored by TLC(9/1 DCM/MeOH). Upon completion, the mixture was acidified carefully topH 2 with concentrated HCl and then concentrated in vacuo to remove theTHF. The resulting aqueous layer was washed twice with Et₂O and thecombined organic layers were washed once with brine. The organic layerwas then dried over MgSO₄, filtered and concentrated in vacuo. Thebenzoic acid t-butyl ester was used without further purification.

1 equivalent of 3-methoxybenzonitrile was placed in a Parr bottle withEtOH, 0.02 equivalents of HCl and 10% (w/w) of 10% Pd on carbon. Thevessel was placed in the Parr shaker, charged with 50 psi H2, and shakenfor 12 hours. The reaction filtered through a pad of celite and diluted1:10 with Et₂O. Upon standing over night, fine white needles form. Theproduct was filtered, washed with Et₂O and dried in vacuo. The resultingamine hydrochloride salt was then used with out further purification.

3 equivalents of the benzoic acid t-butyl ester was coupled to 1equivalent of the amine hydrochloride salt using 3 equivalents EDC, 1equivalent of Hobt and 3 equivalents of DIPEA in DMA. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasconcentrated in vacuo. The resulting oil was re suspended in Et₂O andwashed twice with 0.1 N H₂SO₄, twice with saturated NaHCO₃, and oncewith brine. The organic layer was then dried over MgSO₄, filtered andconcentrated in vacuo. The product was then purified on silica get using5% methanol in DCM as eluent to provide pure t-butyl ester.

The t-butyl ester was dissolved in a solution of TFA in DCM (1:1). After20 minutes, the reaction was concentrated in vacuo. The resulting oilwas dissolved in toluene and then concentrated in vacuo twice.

The resulting compound was dissolved in DCM and cooled to −5° C. in anice/acetone bath under nitrogen. 2 equivalents of BBr₃ were added dropwise as a solution in DCM over 30 minutes. The reaction was warmed toroom temperature and stirred until complete by TLC (DCM/2% HOAc/2%MeOH). The solution was poured onto ice, and the ice was allowed tomelt. The mixture was then partitioned twice with EtOAc and the combinedorganic layers were dried over MgSO₄. The filtrate was then passed overa plug of silica gel and concentrated in vacuo to afford pure benzoicacid.

1 equivalent of the benzoic acid, 2 equivalents of commerciallyavailable □-Boc-diaminopropionic acid methyl ester, 2 equivalents ofEDC, 1 equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure Boc methyl ester.

1 equivalent of commercially available nipecotic acid was dissolved in a3:2 THF/H₂O solution. 1.1 equivalents of solid NaHCO₃ and 1.1equivalents of Boc₂O were added and the mixture was stirred overnight.The reaction was concentrated to remove the THF, and the resultingaqueous layer was partitioned with hexanes. The aqueous layer was thenacidified to pH 2 with 1N HCl and then partitioned twice with EtOAc. Thecombined organic layers were dried over MgSO₄ and concentrated in vacuo.The resulting Boc protected nipecotic acid was used without furtherpurification.

The Boc methyl ester was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then re concentrated in vacuo. 1equivalent of this amine, 2 equivalents of resulting Boc protectednipecotic acid, 2 equivalents of EDC, 1 equivalent of Hobt and 3equivalents of DIPEA were dissolved DMA. The reaction was stirred atroom temperature and monitored by TLC (9/1 DCM/MeOH). Upon completion,the mixture was concentrated in vacuo. The resulting oil was resuspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure product.

This Boc protected product was dissolved in a solution of TFA in DCM(1:1). After 20 minutes, the reaction was concentrated in vacuo. Theresulting oil was dissolved in toluene and then concentrated in vacuotwice to provide pure amine. 1 equivalent of this amine, 2 equivalentsof the appropriate commercially available acid (example 29; propionicacid; example 30, acetic acid), 2 equivalents of EDC, 1 equivalent ofHobt and 3 equivalents of DIPEA were dissolved DMA. The reaction wasstirred at room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure product.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The resulting acid was then purifiedby reverse phase HPLC, verified by electrospray mass spectrometry andlyophilized to a powder.

Example 7 Synthesis of Compounds 32-34

1 equivalent of dimethyl 2-chloroterephthalic acid was dissolved in DCMand cooled to −5° C. in an ice/acetone bath under nitrogen. 1 equivalentof BBr₃ was added drop wise as a solution in DCM over 30 minutes. Thereaction was warmed to room temperature and stirred until complete byTLC (DCM/2% HOAc/2% MeOH). The solution was poured onto ice, and the icewas allowed to melt. The mixture was then partitioned with EtOAc andconcentrated in vacuo. This product was dissolved in H₂O with theaddition of saturated NaHCO₃ until the pH remained above 8. Thissolution was partitioned one time with and equal volume of DCM to removeunreacted diester. The basic solution was acidified at 0° C. withconcentrated HCl to pH=1-1.5, and precipitate was extracted twice withequal volumes of EtOAc. The oraganics were partitioned once with brineand dried over MgSO₄, filtered and concentrated in vacuo. Product was7:1 of the correct regioisomer by HPLC.

The monoester was dissolved in DCM and transferred to a pre-weighed Parrflask containing a stirring bar. The flask was cooled to −5° C. with adry ice/alcohol bath under nitrogen. Once cool, ˜30 equivalents ofisobutylene was pumped into solution with stirring. 2.1 equivalents ofconcentrated sulfuric acid was added and the flask was sealed with awired rubber stopper and allowed to warm to room temperature withstirring. The solution was stirred until clarification (1-2 days). Oncethe solution was clear, it was cooled to 0° C. in an ice bath. Thestopper was removed and the excess isobutylene was blown off withnitrogen bubbling. Saturated NaHCO₃ was added to neutralize the acid andthe mixture was concentrated in vacuo until no DCM remained. Thesolution was then partitioned into EtOAc. The oraganics were partitionedtwice with dilute HCl, twice with saturated NaHCO₃, once with brine,dried over MgSO₄, filtered and concentrated in vacuo. The resultingproduct was used with no further purification.

1 equivalent of the methyl ester was dissolved in THF/H₂O (3/1) and 3equivalents of LiOH.H₂O was added. The reaction was monitored by TLC(9/1 DCM/MeOH). Upon completion, the mixture was acidified carefully topH 2 with concentrated HCl and then concentrated in vacuo to remove theTHF. The resulting aqueous layer was washed twice with Et₂O and thecombined organic layers were washed once with brine. The organic layerwas then dried over MgSO₄, filtered and concentrated in vacuo. Thebenzoic acid t-butyl ester was used without further purification.

1 equivalent of 3-methoxybenzonitrile was placed in a Parr bottle withEtOH, 0.02 equivalents of HCl and 10% (w/w) of 10% Pd on carbon. Thevessel was placed in the Parr shaker, charged with 50 psi H2, and shakenfor 12 hours. The reaction filtered through a pad of celite and diluted1:10 with Et₂O. Upon standing over night, fine white needles form. Theproduct was filtered, washed with Et₂O and dried in vacuo. The resultingamine hydrochloride salt was then used with out further purification.

3 equivalents of the benzoic acid t-butyl ester was coupled to 1equivalent of the amine hydrochloride salt using 3 equivalents EDC, 1equivalent of Hobt and 3 equivalents of DIPEA in DMA. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasconcentrated in vacuo. The resulting oil was re suspended in Et₂O andwashed twice with 0.1 N H₂SO₄, twice with saturated NaHCO₃, and oncewith brine. The organic layer was then dried over MgSO₄, filtered andconcentrated in vacuo. The product was then purified on silica get using5% methanol in DCM as eluent to provide pure t-butyl ester.

The t-butyl ester was dissolved in a solution of TFA in DCM (1:1). After20 minutes, the reaction was concentrated in vacuo. The resulting oilwas dissolved in toluene and then concentrated in vacuo twice.

The resulting compound was dissolved in DCM and cooled to −5° C. in anice/acetone bath under nitrogen. 2 equivalents of BBr₃ were added dropwise as a solution in DCM over 30 minutes. The reaction was warmed toroom temperature and stirred until complete by TLC (DCM/2% HOAc/2%MeOH). The solution was poured onto ice, and the ice was allowed tomelt. The mixture was then partitioned twice with EtOAc and the combinedorganic layers were dried over MgSO₄. The filtrate was then passed overa plug of silica gel and concentrated in vacuo to afford pure benzoicacid.

1 equivalent of the benzoic acid, 2 equivalents of commerciallyavailable □-Boc-diaminopropionic acid methyl ester, 2 equivalents ofEDC, 1 equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure Boc methyl ester.

1 equivalent of commercially available isonipecotic acid was dissolvedin a 3:2 THF/H₂O solution. 1.1 equivalents of solid NaHCO₃ and 1.1equivalents of Boc₂O were added and the mixture was stirred overnight.The reaction was concentrated to remove the THF, and the resultingaqueous layer was partitioned with hexanes. The aqueous layer was thenacidified to pH 2 with 1N HCl and then partitioned twice with EtOAc. Thecombined organic layers were dried over MgSO₄ and concentrated in vacuo.The resulting Boc protected isonipecotic acid was used without furtherpurification.

The Boc methyl ester was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then re concentrated in vacuo. 1equivalent of this amine, 2 equivalents of resulting Boc protectedisonipecotic acid, 2 equivalents of EDC, 1 equivalent of Hobt and 3equivalents of DIPEA were dissolved DMA. The reaction was stirred atroom temperature and monitored by TLC (9/1 DCM/MeOH). Upon completion,the mixture was concentrated in vacuo. The resulting oil was resuspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure product.

This Boc protected product was dissolved in a solution of TFA in DCM(1:1). After 20 minutes, the reaction was concentrated in vacuo. Theresulting oil was dissolved in toluene and then concentrated in vacuotwice to provide pure amine. 1 equivalent of this amine, 2 equivalentsof the appropriate commercially available acid (example 32; propionicacid; example 33, butyric acid; example 34, acetic acid), 2 equivalentsof EDC, 1 equivalent of Hobt and 3 equivalents of DIPEA were dissolvedDMA. The reaction was stirred at room temperature and monitored by TLC(9/1 DCM/MeOH). Upon completion, the mixture was concentrated in vacuo.The resulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure product.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O were added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The resulting acid was then purifiedby reverse phase HPLC, verified by electrospray mass spectrometry andlyophilized to a powder.

Example 8 Synthesis of Compounds 36

1 equivalent of 2,6-Dichloro-4-methyl phenol was dissolved in DCMcontaining 2.6 equivalents of 2,6-lutidine and the mixture was cooled to−78° C. After adding 1.25 equivalents of triflic anhydride the stirringreaction was allowed to warm to room temperature overnight. The reactionwas then concentrated, and the residue was partitioned between Et₂O andH₂O. The aqueous layer was extracted with Et₂O and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo. The residue waspurified by silica gel flash chromatography (9:1 hexane/Et₂O) to providethe pure triflate.

To a stirring solution of 1 equivalent of the triflate in a 2/1 mixtureof DMF/MeOH was added 0.15 equivalents of1,3-bis(diphenylphosphino)-propane and 2.5 equivalents of TEA. Carbonmonoxide gas was bubbled through this solution for 15 minutes, then 0.15equivalents of Pd(OAc)2 was added and the reaction was stirred at 70° C.for 5-7 hours under an atmosphere of CO (using a balloon filled withCO). The reaction was then concentrated in vacuo, and the residue waspartitioned between Et₂O and H₂O. The aqueous layer was extracted twicewith Et₂O and the combined organic layers were dried over MgSO₄,filtered through a plug of silica gel and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (9:1:0.02hexane/DCM/Et₂O) to provide the pure tolyl methyl ester.

1 equivalent of the tolyl methyl ester was dissolved in acetic anhydrideand HOAc, then cooled in an ice-salt bath (−5° C.) before concentratedH₂SO₄ was added. A solution of CrO₃ (2.6 equivalents) in aceticanhydride and HOAc was added drop wise and the reaction was stirred for3.5 hours at −5° C. The reaction was poured into ice H₂O and stirred for30 min. The mixture was extracted three times with ethyl ether. Thecombined organic layers were washed with saturated NaHCO₃ and brine,then dried over MgSO₄ and concentrated in vacuo to an oil. Toluene wasadded to the oil and the solution concentrated in vacuo again. This wasrepeated to obtain a crystalline solid. The solid was dissolved inmethanol and concentrated HCl and heated at reflux for 12 hours. Thereaction was concentrated in vacuo and the residue was purified bysilica gel flash chromatography (9:1 hexane/Et₂O) to provide the purealdehyde.

A solution of 1 equivalent of the aldehyde in THF was cooled to −78° C.and 1.1 equivalents of 0.5M ethynylmagnesium bromide/THF was added.After stirring the reaction at room temperature for 3 hours, it wasdiluted with Et₂O and washed twice with 10% citric acid. The combinedaqueous layers were back-extracted once with Et₂O. The combined organiclayers were washed twice with saturated aqueous NaHCO₃, dried over MgSO₄and concentrated in vacuo. The residue was purified by silica gel flashchromatography (4:1 to 3:2 hexane/Et₂O) to provide the pure alkyne.

1 equivalent of 3-Iodophenol, 2.2 equivalents oft-butyldimethyl silylchloride and 3 equivalents of imidizole were dissolved in DMF andstirred at room temperature. The reaction was monitored by TLC (9/1DCM/MeOH). Upon reaction completion, the mixture was concentrated invacuo. The resulting oil was re suspended in Et₂O and washed twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The product was thenused with out further purification.

1 equivalent of the silyl iodide was dissolved in EtOAc and the solutionwas degassed by passing N2 through a pipette and into the solution for10 minutes. 1.25 equivalents of the alkyne was added, followed by 0.02equivalents of dichlorobis(triphenylphosphine)-palladium-(II), 0.04equivalents of CuI and 5 equivalents TEA. The reaction was stirred for14 hours, diluted with EtOAc, washed twice with 5% Na₂.EDTA, brine andthen dried over MgSO₄ and concentrated in vacuo. The residue waspurified by silica gel flash chromatography (gradient elution, usingEt₂O to EtOAc) to provide the pure aryl alkyne.

1 equivalent of the aryl alkyne was dissolved in MeOH and the solutionwas degassed by passing N2 through a pipette and into the solution for10 minutes. The 5% Rh/Al₂O₃ was added, one balloon-full of hydrogen waspassed through the solution, and the reaction was stirred under anatmosphere of H₂ (using a balloon) for 7 hours, after which the reactionwas filtered through a pad of celite and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (gradientelution, using Et₂O to EtOAc) to provide the pure product.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents ofBoc-L-thiazolidine-4-carboxylic acid, 2 equivalents of EDC, 1 equivalentof Hobt and 3 equivalents of DIPEA were dissolved DMA. The reaction wasstirred at room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The Boc, silyl residue was dissolvedin a solution of TFA in DCM (1:1) with 3 equivalents of TBAF. After 20minutes, the reaction was concentrated in vacuo. The resulting oil wasdissolved in toluene and then reconcentrated in vacuo. The resultingacid was then purified by reverse phase HPLC, verified by electrospraymass spectrometry and lyophilized to a powder.

Example 9 Synthesis of Compounds 37

1 equivalent of 2,6-Dichloro-4-methyl phenol was dissolved in DCMcontaining 2.6 equivalents of 2,6-lutidine and the mixture was cooled to−78° C. After adding 1.25 equivalents of triflic anhydride the stirringreaction was allowed to warm to room temperature overnight. The reactionwas then concentrated, and the residue was partitioned between Et₂O andH₂O. The aqueous layer was extracted with Et₂O and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo. The residue waspurified by silica gel flash chromatography (9:1 hexane/Et₂O) to providethe pure triflate.

To a stirring solution of 1 equivalent of the triflate in a 2/1 mixtureof DMF/MeOH was added 0.15 equivalents of1,3-bis(diphenylphosphino)-propane and 2.5 equivalents of TEA. Carbonmonoxide gas was bubbled through this solution for 15 minutes, then 0.15equivalents of Pd(OAc)2 was added and the reaction was stirred at 70° C.for 5-7 hours under an atmosphere of CO (using a balloon filled withCO). The reaction was then concentrated in vacuo, and the residue waspartitioned between Et₂O and H₂O. The aqueous layer was extracted twicewith Et₂O and the combined organic layers were dried over MgSO₄,filtered through a plug of silica gel and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (9:1:0.02hexane/DCM/Et₂O) to provide the pure tolyl methyl ester.

1 equivalent of the tolyl methyl ester was dissolved in acetic anhydrideand HOAc, then cooled in an ice-salt bath (−5° C.) before concentratedH₂SO₄ was added. A solution of CrO₃ (2.6 equivalents) in aceticanhydride and HOAc was added drop wise and the reaction was stirred for3.5 hours at −5° C. The reaction was poured into ice H₂O and stirred for30 min. The mixture was extracted three times with ethyl ether. Thecombined organic layers were washed with saturated NaHCO₃ and brine,then dried over MgSO₄ and concentrated in vacuo to an oil. Toluene wasadded to the oil and the solution concentrated in vacuo again. This wasrepeated to obtain a crystalline solid. The solid was dissolved inmethanol and concentrated HCl and heated at reflux for 12 hours. Thereaction was concentrated in vacuo and the residue was purified bysilica gel flash chromatography (9:1 hexane/Et₂O) to provide the purealdehyde.

A solution of 1 equivalent of the aldehyde in THF was cooled to −78° C.and 1.1 equivalents of 0.5M ethynylmagnesium bromide/THF was added.After stirring the reaction at room temperature for 3 hours, it wasdiluted with Et₂O and washed twice with 10% citric acid. The combinedaqueous layers were back-extracted once with Et₂O. The combined organiclayers were washed twice with saturated aqueous NaHCO₃, dried over MgSO₄and concentrated in vacuo. The residue was purified by silica gel flashchromatography (4:1 to 3:2 hexane/Et₂O) to provide the pure alkyne.

1 equivalent of 1-chloro-3-iodobenzene was dissolved in EtOAc and thesolution was degassed by passing N2 through a pipette and into thesolution for 10 minutes. 1.25 equivalents of the alkyne was added,followed by 0.02 equivalents ofdichlorobis(triphenylphosphine)palladium-(II), 0.04 equivalents of CuIand 5 equivalents TEA. The reaction was stirred for 14 hours, dilutedwith EtOAc, washed twice with 5% Na₂.EDTA, brine and then dried overMgSO₄ and concentrated in vacuo. The residue was purified by silica gelflash chromatography (gradient elution, using Et₂O to EtOAc) to providethe pure aryl alkyne.

1 equivalent of the aryl alkyne was dissolved in MeOH and the solutionwas degassed by passing N2 through a pipette and into the solution for10 minutes. The 5% Rh/Al₂O₃ was added, one balloon-full of hydrogen waspassed through the solution, and the reaction was stirred under anatmosphere of H₂ (using a balloon) for 7 hours, after which the reactionwas filtered through a pad of celite and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (gradientelution, using Et₂O to EtOAc) to provide the pure product.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

1 equivalent of commercially available D-hydroxy proline was dissolvedin a 3:2 THF/H₂O solution. 1.1 equivalents of solid NaHCO₃ and 1.1equivalents of Boc₂O were added and the mixture was stirred overnight.The reaction was concentrated to remove the THF, and the resultingaqueous layer was partitioned with hexanes. The aqueous layer was thenacidified to pH 2 with IN HCl and then partitioned twice with EtOAc. Thecombined organic layers were dried over MgSO₄ and concentrated in vacuo.The resulting N-Boc-D-hydroxy proline was used without furtherpurification.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. 1equivalent of this amine, 2 equivalents of Boc-D-hydroxy proline, 2equivalents of EDC, 1 equivalent of Hobt and 3 equivalents of DIPEA weredissolved DMA. The reaction was stirred at room temperature andmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasconcentrated in vacuo. The resulting oil was re suspended in Et₂O andwashed twice with 0.1 N H₂SO₄, twice with saturated NaHCO₃, and oncewith brine. The organic layer was then dried over MgSO₄, filtered andconcentrated in vacuo. The residue was then purified on silica get using5% methanol in DCM as eluent to provide pure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The Boc, silyl residue was dissolvedin a solution of TFA in DCM (1:1). After 20 minutes, the reaction wasconcentrated in vacuo. The resulting oil was dissolved in toluene andthen reconcentrated in vacuo. The resulting acid was then purified byreverse phase HPLC, verified by electrospray mass spectrometry andlyophilized to a powder.

Example 10 Synthesis of Compound 35

A round bottom flask was equipped with an efficient overhead stirrer andcharged with concentrated H₂SO₄ (2.7×volume of H₂O) and H₂O and cooledto ˜−5° C. with an ethanol/ice bath. Once cool, 1 equivalent 2.6dichloro phenol and 1 equivalent of N-(hydroxymethyl)phthalimide wereadded with vigorous stirring. The reaction was kept cool for 4 hours andthen allowed to warm to room temperature overnight with constantstirring. The reaction generally proceeds to a point where there wasjust a solid in the round bottom flask. At this point EtOAc and H₂O wereadded and stirred into the solid. Large chunks were broken up and thenthe precipitate was filtered and washed with more EtOAc and H₂O. Theproduct was then used without further purification after dryingovernight under vacuum.

1 equivalent of the dry product and methanol (22.5 ml×#g of startingmaterial) was added to a round bottom flask equipped with a H₂Ocondenser and stirring bar. 1.2 equivalents of hydrazine mono hydratewas added and the mixture refluxed for 4 hours. After cooling to roomtemperature, concentrated HCl (4.5 ml×#g of starting material) wascarefully added. Upon completion of the addition, the mixture wasrefluxed overnight (>8 hours). The reaction was cooled to 0° C. and theprecipitated by-product was removed by filtration. The filtrate was thenconcentrated in vacuo.

The crude amine residue was dissolved in a 3:2 THF/H₂O solution. 1.1equivalents of solid NaHCO₃ and 1.1 equivalents of Boc₂O were added andthe mixture was stirred overnight. The reaction was concentrated, andthe residue was partitioned between H₂O and Et₂O. The aqueous layer wasextracted with Et₂O and the combined organic layers were dried overMgSO₄ and concentrated in vacuo to a solid. Recrystallization from hotmethanol and H₂O provided pure product.

1 equivalent of the Boc protected amine and 1.5 equivalents of2,6-lutidine was dissolved, with mild heating if necessary, in DCM in around bottom flask. Once the starting material has completely dissolved,the mixture was cooled to −78° C. under N₂ with a dry ice ethanol bath.Once cool, 2.5 equivalents of triflic anhydride was added and thereaction was allowed to slowly come to room temperature with stirring.The reaction was monitored by TLC and was generally done in 4 hours.Upon completion, the reaction was concentrated in vacuo and the residuepartitioned between EtOAc and H₂O. The organic layer was washed twicewith 0.1N H₂SO₄, twice with saturated NaHCO₃, once with brine, driedover MgSO₄ and concentrated in vacuo. The residue was then purified onsilica gel using DCM as eluent to provide pure triflate.

1 equivalent of triflate was dissolved in DMF and MeOH in the glassinsert of a high pressure Parr bomb. The starting material was thendegassed while stirring with CO for 10 minutes. 0.15 equivalentspalladium(II) acetate and 0.15 equivalents of 1,3-bis(diphenylphosphino)propane were then added and the mixture was then degassed while stirringwith CO for another 10 minutes at which time 2.5 equivalents ofdiisopropyl ethyl amine was added. After properly assembling the bomb,it was charged with 300 psi CO gas and heated to 70° C. with stirringovernight. The bomb was then cooled and vented. The mixture wastransferred to a round bottom flask and concentrated in vacuo. Theresidue was then purified on silica gel using DCM with 1% acetone and 1%TEA as eluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then reconcentrated in vacuo. The TFAsalt of the amine was dissolved in Et₂O and washed twice with a 10%solution of K₂CO₃ in H₂O and once with brine. The organic layer was thendried over MgSO₄, filtered and concentrated in vacuo.

1 equivalent of the free based amine, 3 equivalents of furylacrylicacid, 3 equivalents of EDC and 1 equivalent of Hobt were dissolved DMA.The reaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

2.3 equivalents of lithium iodide was added to 1 equivalent of themethyl ester in pyridine, and the mixture heated at reflux for 8 hours.The reaction was concentrated in vacuo and the residue was partitionedbetween EtOAc and 1N HCl. The aqueous layer was extracted three timeswith EtOAc, and the combined organic layers were washed with 1M NaHCO₃,dried over MgSO₄ and concentrated in vacuo. The residue was dissolved inNMM and the solution concentrated in vacuo. The residue was taken up inDCM and then washed three times with 1N HCl. The organic layer was driedover MgSO₄ and concentrated in vacuo to provide the benzoic acid in highenough purity to be used without further purification.

1 equivalent of the acid, 2 equivalents of commercially availableβ-Boc-diaminopropionic acid methyl ester, 2 equivalents of EDC, 1equivalent of Hobt and 3 equivalents of DIPEA were dissolved DMA. Thereaction was stirred at room temperature and monitored by TLC (9/1DCM/MeOH). Upon completion, the mixture was concentrated in vacuo. Theresulting oil was re suspended in Et₂O and washed twice with 0.1 NH₂SO₄, twice with saturated NaHCO₃, and once with brine. The organiclayer was then dried over MgSO₄, filtered and concentrated in vacuo. Theresidue was then purified on silica get using 5% methanol in DCM aseluent to provide pure methyl ester.

The Boc protected amine was dissolved in a solution of TFA in DCM (1:1).After 20 minutes, the reaction was concentrated in vacuo. The resultingoil was dissolved in toluene and then re concentrated in vacuo.

To 1 equivalent of this amine was added 1.05 equivalents of methyliodide and 2.1 equivalents potassium carbonate in DMF. The reaction wasstirred at room temperature and followed by TLC (9/1 DCM/MeOH). Uponcompletion of the reaction, it was diluted with EtOAc and H₂O. Theaqueous layer was partitioned again with EtOAc and the combined organiclayers washed with brine, dried over MgSO₄ and concentrated in vacuo.

1 equivalent of this amine, 2 equivalents ofBoc-L-thiazolidine-4-carboxylic acid, 2 equivalents of EDC, 1 equivalentof Hobt and 3 equivalents of DIPEA were dissolved DMA. The reaction wasstirred at room temperature and monitored by TLC (9/1 DCM/MeOH). Uponcompletion, the mixture was concentrated in vacuo. The resulting oil wasre suspended in Et₂O and washed twice with 0.1 N H₂SO₄, twice withsaturated NaHCO₃, and once with brine. The organic layer was then driedover MgSO₄, filtered and concentrated in vacuo. The residue was thenpurified on silica get using 5% methanol in DCM as eluent to providepure methyl ester.

1 equivalent of the resultant methyl ester was dissolved in THF/H₂O(3/1) and 3 equivalents of LiOH.H₂O was added. The reaction wasmonitored by TLC (9/1 DCM/MeOH). Upon completion, the mixture wasacidified to pH 2 with 1M HCl and then concentrated in vacuo. Theresulting solid was re suspended in Et₂O and washed twice with 0.1 M HCland once with brine. The organic layer was then dried over MgSO₄,filtered and concentrated in vacuo. The residue was dissolved in asolution of TFA in DCM (1:1). After 20 minutes, the reaction wasconcentrated in vacuo. The resulting oil was dissolved in toluene andthen re concentrated in vacuo. The resulting acid was then purified byreverse phase HPLC, verified by electrospray mass spectrometry andlyophilized to a powder.

Example 11 PLM2 Antibody Capture LFA-1:ICAM-1 Assay

A non-function blocking monoclonal antibody against human CD18, PLM-2(as described by Hildreth, et al., Molecular Immunology, Vol. 26, No. 9,pp. 883-895, 1989), is diluted to 5 μg/ml in PBS and 96-wellflat-bottomed plates are coated with 100 μl/well overnight at 4° C. Theplates are blocked with 0.5% BSA in assay buffer (0.02M Hepes, 0.15MNaCl, and 1 mM MnCl₂) 1 h at room temperature. Plates are washed with 50mM Tris pH 7.5, 0.1M NaCl, 0.05% Tween 20 and 1 mM MnCl2. Purifiedfull-length recombinant human LFA-1 protein is diluted to 2 μg/ml inassay buffer and 100 μl/well is added to plates and incubated 1 h at 37°C. Plates are washed 3×. 50 μl/well inhibitors, appropriately diluted inassay buffer, are added to a 233 final concentration and incubated for30′ at 37° C. 50 μl/well of purified recombinant human 5 domain ICAM-Ig,diluted to 161 ng/ml (for a final concentration of 80 ng/ml) in assaybuffer, is added and incubated 2 h at 37° C. Plates are washed and boundICAM-Ig is detected with Goat anti-HuIgG(Fc)-HRP for 1 h at roomtemperature. Plates are washed and developed with 100 μl/well TMBsubstrate for 5-10′ at room temperature. Colorimetric development isstopped with 100 μl/well 1M H₃PO₄ and read at 450 nM on a platereader.Results of the PLM2 assay are shown in tables 1-4 below.

Example 12 Serum/Plasma Protein Binding

Binding of test compounds was performed according to proceduresdescribed in Borga et al (Journal of Pharmacokinetics &Biopharmaceutics, 1997, 25(1):63-77) and Godolphin et al (Therapeuticdrug monitoring, 1983, 5:319-23). Duplicate samples of 10 μl of testcompound stock solution (1 μg/μL) was spiked into 1 mL of either bufferor serum/plasma adjusted to pH 7.4 using CO₂ at room temperature.Samples were equilibrated by incubating vials in a water bath withshaker at 37° C. for 15 minutes. 200 μl of the buffer spiked sample wassaved as prefiltrate. 800 μl of buffer spiked samples and 1 ml of serumspiked samples were centrifuged at 1500 g, 37° C., for 30 minutes in aCentrifree ultrafiltration device (Amicon Inc.). Pre and post-filtrateswere then analyzed by LC/MS-MS and percent binding of test compound toserum/plasma protein was determined from the post and prefiltratesaccounting for any non-specific binding determined from the buffercontrol.

Compounds of the invention incorporating a non-aromatic ring atsubstituent Cy surprisingly exhibit low serum plasma protein bindingcharacteristics which is advantageous for maintaining therapeuticallyrelevant serum levels. As illustrated in tables 1-4, reference compounds(ref) having an aromatic ring at substituent Cy consistently show higher% plasma protein binding compared to the equivalent compound of theinvention having a non-aromatic ring.

TABLE 1 LFA-1 % PLM2 Mac-1 plasma cmpd IC₅₀ IC₅₀ protein no. (μM) (μM)binding structure ref 0.071 98.3

 4 0.004 82.9

 5 0.008 83.1

35 0.009 51.36

17 0.003 84.61

10 0.003 65.91

12 0.002 79.48

13 0.004 77.58

14 0.002 72.60

41 0.003 84.83

44 0.002 82.97

TABLE 2 LFA-1 % PLM2 Mac-1 plasma cmpd IC₅₀ IC₅₀ protein no. (μM) (μM)binding structure ref 0.005 98.12

ref 0.004 161 99.5

 6 0.007 2509 95.43

15 0.004 92.51

36 0.002 65 92.84

37 35.54 93.19

38 0.012 7609 93.29

40 0.002 1427 96.93

42 0.003 91.4

TABLE 3 LFA-1 % PLM2 Mac-1 plasma cmpd IC₅₀ IC₅₀ protein no. (μM) (μM)binding structure ref 0.015 99.4

9 0.002 77.17

3 0.011 80.8

TABLE 4 LFA-1 % PLM2 Mac-1 plasma cmpd IC₅₀ IC₅₀ protein no. (μM) (μM)binding structure ref 99.2

ref 0.002 1683 99.70

51 0.005 2362 92.8

We claim:
 1. A compound of formula (I)

wherein Cy is a non-aromatic heterocycle optionally substituted withhydroxyl, mercapto, thioalkyl, halogen, oxo, thio, amino, aminoalkyl,amidine, guanidine, nitro, alkyl, alkoxy or acyl; X is—CH₂—NR₆—[divalent hydrocarbon chain]—wherein said divalent hydrocarbonchain is optionally substituted with hydroxyl, mercapto, halogen, amino,aminoalkyl, nitro, oxo or thio; Y is a heterocycle optionallysubstituted with hydroxyl, mercapto, halogen, oxo, thio, thioalkyl,amino, aminoalkyl, carbocycle or heterocycle ring, hydrocarbon, ahalo-substituted hydrocarbon, amino, amidine, guanidine, cyano, nitro,alkoxy or acyl; L is —[divalent hydrocarbon chain]—NR₆—CH₂— wherein saiddivalent hydrocarbon chain is optionally substituted with hydroxyl,halogen, oxo or thio and R₆ is H or alkyl; R₁ is H, OH, amino,O-carbocycle or alkoxy optionally substituted with amino, a carbocycleor heterocycle; R₂₋₅ are independently H, hydroxyl, mercapto, halogen,cyano, amino, amidine, guanidine, nitro or alkoxy; R₆ is H or ahydrocarbon chain optionally substituted with a carbocycle or aheterocycle; and salts, solvates and hydrates thereof.
 2. A compoundaccording to claim 1, wherein Cy is a 5- or 6-member non-aromaticheterocycle optionally substituted with hydroxyl, mercapto, thioalkylhalogen, oxo, thio, amino, aminoalkyl, amidine, guanidine, nitro, alkyl,alkoxy or acyl.
 3. A compound according to claim 2, wherein saidheterocycle comprises one or two heteroatoms and is optionallysubstituted with hydroxyl, oxo, mercapto, thio, alkyl or alkanoyl.
 4. Acompound according to claim 3, wherein said heterocycle is selected fromthe group consisting of piperidine, piperazine, morpholine,tetrahydrofuran, tetrahydrothiophene, oxazolidine,cyclopropa-pyrrolidine and thiazolidine optionally substituted withhydroxy, oxo, mercapto, thio, alkyl or alkanoyl.
 5. A compound accordingto claim 4, wherein said heterocycle is selected from the groupconsisting of piperidine, piperazine, morpholine, tetrahydrofuran,tetrahydrothiophene, oxazolidine, thiazolidine optionally substitutedwith hydroxy, oxo, mercapto, thio, alkyl or alkanoyl.
 6. A compoundaccording to claim 1, wherein X is —CH₂—NR₆—C(O)— wherein the carbonyl—C(O)— portion thereof is covalently bound to Cy and R₆ is H or alkyl.7. A compound according to claim 1, wherein Y is a heterocycleoptionally substituted with hydroxyl or halogen.
 8. A compound accordingto claim 7, wherein Y is furan-2-yl or thiophene-2-yl.
 9. A compoundaccording to claim 1, wherein L is —CH═CH—C(O)—NR₆—CH₂—, or—C(O)—NR₆—CH₂—; wherein each R₆ is independently H or alkyl.
 10. Acompound according to claim 9, wherein R₁ is H, OH, amino, O-carbocycleor alkoxy optionally substituted with a carbocycle.
 11. A compoundaccording to claim 10, wherein R₁ is H or C₁₋₄ alkyloxy.
 12. A compoundaccording to claim 1, wherein at least one of R₂ and R₃ is halogen andthe other is H or halogen.
 13. A compound according to claim 12, whereinR₂ and R₃ are both Cl.
 14. A compound according to claim 13, wherein R₄and R₅ are both H.
 15. A pharmaceutical composition comprising acompound according to claim 1 with a pharmaceutically acceptableadjuvant, diluent or carrier.
 16. A method of inhibiting binding of aLFA-1 to a protein ligand comprising contacting LFA-1 with a compound ofclaim
 1. 17. A method of treating a disease or condition mediated byLFA-1 in a mammal comprising administering to said mammal an effectiveamount of a compound according to claim
 1. 18. A method according toclaim 17, wherein said disease or condition is arthritis, psoriasis,organ transplant rejection, asthma, and inflammatory bowel disease. 19.A method according to claim 17, wherein said disease or condition is aninflammatory disease or condition.
 20. A compound of formula (Ib)

wherein Cy is a non-aromatic heterocycle optionally substituted withhydroxyl, mercapto, thioalkyl, halogen, oxo, thio, amino, aminoalkyl,amidine, guanidine, nitro, alkyl, alkoxy or acyl; Y is a heterocycleoptionally substituted with hydroxyl, mercapto, halogen, oxo, thio,thioalkyl, amino, aminoalkyl, carbocycle or heterocycle ring,hydrocarbon, a halo-substituted hydrocarbon, amino, amidine, guanidine,cyano, nitro, alkoxy or acyl; L is a divalent hydrocarbon chainoptionally substituted with hydroxyl, halogen, oxo or thio; R₁ is H, OH,amino, O-carbocycle or alkoxy optionally substituted with amino, acarbocycle or heterocycle; R₂ and R₃ are independently H, hydroxyl,mercapto, halogen, cyano, amino, amidine, guanidine, nitro or alkoxy; R₆is H or a hydrocarbon chain optionally substituted with a carbocycle ora heterocycle; and salts, solvates and hydrates thereof.
 21. A compoundof formula (If)

wherein Cy is a non-aromatic heterocycle optionally substituted withhydroxyl, mercapto, thioalkyl, halogen, oxo, thio, amino, aminoalkyl,amidine, guanidine, nitro, alkyl, alkoxy or acyl; Y is a heterocycleoptionally substituted with hydroxyl, mercapto, halogen, oxo, thio,thioalkyl, amino, aminoalkyl, carbocycle or heterocycle ring,hydrocarbon, a halo-substituted hydrocarbon, amino, amidine, guanidine,cyano, nitro, alkoxy or acyl; R₁ is H, OH, amino, O-carbocycle or alkoxyoptionally substituted with amino, a carbocycle or heterocycle; R₂ andR₃ are independently H, hydroxyl, mercapto, halogen, cyano, amino,amidine, guanidine, nitro or alkoxy; R₆ is H or a hydrocarbon chainoptionally substituted with a carbocycle or a heterocycle; and salts,solvates and hydrates thereof.